Macrolide antiinfective agents

ABSTRACT

The invention is directed towards antibacterial compounds. The invention concerns macrolide antibiotics useful as antiinfective agents.

[0001] This application claims priority under 35 U.S.C. §119 from U.S.provisional patent application Serial Nos. 60/173,805 filed Dec. 30,1999, and 60/173,804 filed Dec. 30, 1999 and U.S. utility patentapplication Ser. Nos. 09/551,162 filed Apr. 14, 2000, which in turnclaims priority from U.S. provisional patent application Serial Nos.60/129,729 filed Apr. 16, 1999 and 60/172,154 filed Dec. 17, 1999, and09/550,045 filed Apr. 14, 2000, which in turn claims priority from U.S.provisional patent application Serial Nos. 60/140,175 filed Jun. 18,1999 and 60/172,159 filed Dec. 17, 1999. The contents of theseprovisional and utility applications are relied on and incorporatedherein in their entirety by reference.

TECHNICAL FIELD

[0002] The invention is directed to antibacterial compounds that expandthe repertoire of erythromycin-like antibiotics. More particularly, theinvention concerns macrolide antibiotics containing an erythronolidenucleus modified at least at the substituent at C-13.

BACKGROUND ART

[0003] The increasing number of microbial strains that have acquiredresistance to the currently available known antibiotic compounds isrecognized as a dangerous threat to public health. As the use of suchcompounds has proliferated, so too has the need for expanding theoptions available to treat a wide variety of microbial-based conditions.The need for a larger choice of antimicrobial compounds extends beyondtreatment of human infection and to a need to preserve food and otherperishable commodities. New antibiotics can also be essential forresistant plants and animals as well as to provide resistance tomaterials that otherwise are subject to microbially caused corrosion.

[0004] Thus, there is a clear need for an expanded armament of compoundswhich can provide a multifaceted defense against unwanted microbialactivity.

[0005] WO 98/09978 published Mar. 12, 1998 and incorporated herein byreference discloses modified forms of erythromycin which lack acladinose residue at the 3-position and which are derivatized in variousways in positions 9-12 of the macrolide ring. Similarly, U.S. Pat. No.5,750,510, issued May 12, 1998 and incorporated herein by reference,discloses modified erythromycin derivatives.

[0006] The naturally occurring erythromycins have the structure

Erythromycin R′ R″ A —OH —CH₃ B —H —CH₃ C —OH —H D —H —H

[0007] wherein R′ can be H or OH and R″ can be H or CH₃.

[0008] All of the compounds disclosed in the above-referenced patentdocuments contain an ethyl group at position 13 of the macrolide ring.The present inventors have found that alterations in the substituent atposition 13 results in a large number of compounds with excellentantibacterial activity.

DISCLOSURE OF THE INVENTION

[0009] The invention is directed to erythronolide derivatives thatcontain modifications from the native structure. All of the compounds ofthe invention are modified at least at position 13. In addition, furthermodifications are made at positions 9, 11 and 12, or at positions 12 and13.

[0010] In one embodiment, derivatives contain two fused rings atpositions 9 and 11, and 11-12 of the erythronolide back bone as incompounds (1)-(3) and (1′)-(3′) of the invention. Specifically, thesetwo fused rings include a fused carbamate ring at the 11-12 position,and a fused diamine ring, which is fused to the erythronolide back boneat positions 9 and 11, and also is fused to the carbamate ring.

[0011] In another embodiment, compounds (101)-(103) of the inventioncontain one fused ring at positions 12-13 of the erythronolide backbone. In this embodiment, the erythronolide back bone initially must bemodified at position 13 with a substituent containing a π-bond inpositions α,β to the ring which is then converted to a fused ring atposition 12-13, wherein a 12-hydroxyl group is incorporated into thefused ring, e.g., to form a carbamate ring. Thus, the starter unit forthe erythronolide is in one embodiment an α,β-unsaturated carboxylicacid, which is converted into a thioester, or a diketide thioesterhaving γ,δ-unsaturation.

[0012] Thus, in one aspect, the invention is directed to compounds ofthe formula

[0013] wherein

[0014] R_(a) is substituted or unsubstituted alkyl (1-10C); substitutedor unsubstituted alkenyl (2-10C); substituted or unsubstituted alkynyl(2-10C); substituted or unsubstituted aryl (3-20C); or substituted orunsubstituted arylalkyl (4-20C); or OR_(a) may be replaced by H;

[0015] R_(b) is H or halogen;

[0016] R_(c) is H or a protecting group;

[0017] R_(d) is methyl, unsubstituted alkyl (3-10C); substituted alkyl(1-10C); substituted or unsubstituted alkenyl (2-10C); substituted orunsubstituted alkynyl (2-10C); substituted or unsubstituted aryl(3-20C); substituted or unsubstituted arylalkyl (4-20C); substituted orunsubstituted arylalkenyl (5-20C); substituted or unsubstitutedarylalkynyl (5-20C); substituted or unsubstituted amidoarylalkyl(5-20C); substituted or unsubstituted amidoarylalkenyl (5-20C); orsubstituted or unsubstituted amidoarylalkynyl (5-20C);

[0018] R_(d)′ is H, substituted or unsubstituted alkyl (1-10C);substituted or unsubstituted alkenyl (2-10C); substituted orunsubstituted alkynyl (2-10C); substituted or unsubstituted aryl(4-20C); substituted or unsubstituted arylalkyl (5-20C); substituted orunsubstituted arylalkenyl (5-20C ); substituted or unsubstitutedarylalkynyl; substituted or unsubstituted amidoarylalkyl (5-20C);substituted or unsubstituted amidoarylalkenyl (5-20C); or substituted orunsubstituted amidoarylalkynyl (5-20C);

[0019] R_(e) is H or a protecting group;

[0020] each of A, B, D and E is independently H, substituted orunsubstituted alkyl (1-10C) wherein any pair of said A, B, D and E formsa 3-7-membered ring optionally containing one or more heteroatoms, withthe proviso that at least two of said A, B, D and E must be hydrogen;

[0021] L is methylene or carbonyl;

[0022] T is —O—, —N(R)—, or —N(OR)—, —N(NHCOR)—, —N(N═CHR)—, or —N(NHR)—wherein R is H or R_(a) as defined above, with the proviso that when Lis methylene, T is —O—;

[0023] one of Z and Y is H and the other is OH, protected OH, or amino,mono- or dialkylamino, protected amino, or an amino heterocycle or

[0024] Z and Y together are ═O, ═NOH or a derivatized oxime;

[0025] including any pharmaceutically acceptable salts thereof and anystereoisomeric forms and mixtures of stereoisomeric forms thereof.

[0026] In another aspect, the invention is directed to pharmaceutical orpreservative compositions containing the compounds of formulas (1)-(3),(1′)-(3′) or (101)-(103) and to methods to treat infectious diseases byadministering these compounds or to preserve materials by providingthem.

A BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 shows the post-PKS biosynthesis of erythromycins. Thispathway is employed in the present invention, as shown in FIG. 2.

[0028]FIG. 2 shows a schematic of the synthesis of intermediates for thecompounds of the invention.

[0029]FIG. 3 shows a schematic of the synthesis for intermediates forthe compounds of the invention illustrating the protection and/ordeprotection of the C9, C6, 2′ and 4″ positions.

[0030]FIG. 4 shows a schematic of the synthesis of the compounds of theinvention from the intermediates in FIG. 2.

[0031]FIG. 5 shows a schematic of an alternate synthesis of thecompounds of the invention from the intermediate compound (7).

[0032]FIG. 6 shows a schematic of the synthesis of intermediate cycliccarbonate compounds of compounds (1)-(3) or (1′)-(3′).

[0033]FIGS. 7a and 7 b show a schematic of the synthesis ofintermediates of the compounds of the invention, illustrating theprotection and/or deprotection of the C9, C6 and 2′ positions beforeoxidation or dehydration at the C3 position.

[0034] FIGS. 8 shows schematics of analogous syntheses of compounds ofthe invention.

[0035]FIG. 9 shows the conversion of the hydrogen on the ring nitrogento other substituents.

[0036]FIGS. 10a and 10 b illustrate the oxidation or dehydration at theC3 position after the synthesis of compounds of the invention.

[0037]FIG. 11 illustrates the protection and/or deprotection of the C9and C6 positions and the subsequent conversion of the C6 hydroxyl groupafter formation of the carbamate ring.

[0038]FIG. 12 shows the synthesis of intermediate compounds of formula(6) and their corresponding 10,11-anhydro forms, as described inExamples 7-9.

[0039]FIG. 13 shows the synthesis of intermediate compounds of formula(6) (anhydro form) wherein OR_(a) is replaced by H, as described inExamples 10-12.

[0040]FIG. 14 illustrates the conversion of 15-azidoerythromycin A into15-amidoerythromycins, as described in Example 30.

MODES OF CARRYING OUT THE INVENTION

[0041] The compounds of the invention are conveniently synthesized bycombining synthetic chemical techniques with microbiological processesinvolving genetically engineered microorganisms. Briefly, in a preferredmode of carrying out the invention, a microbial host, preferably a hostwhich does not itself produce a macrolide antibiotic, is provided with arecombinant expression system for the production of modified6-deoxyerythronolide B (6-dEB), as shown in FIG. 1, which expressionsystem will have been altered by a disruption in the catalytic domain ofthe ketosynthase moiety in the first module. For substituents in whichR_(d) is methyl (i.e., in compounds (1)-(3) and (1′)-(3′)) host cellsare used which do not have a disrupted domain of the ketosynthasemoiety. This alteration in the 6-dEB polyketide synthase (PKS) resultsin the inability of this PKS to utilize its native starter unit, andthus permits inclusion of a synthetic diketide thioester for its initialcondensation product in the sequence of reactions leading to modified6-dEB without competition from the diketide that would otherwise,natively, have been produced. Thus, the recombinant host can be provideda synthetic diketide thioester for incorporation into the resultingpolyketide. The incorporation of this diketide into the resultingpolyketide results in a polyketide with a substituent at position 13that may be selected as desired. In intermediate compounds for compounds(101)-(103) of the invention having a fused ring at positions 12-13, thesubstituent at position 13 must be capable of being manipulated in orderto convert it to the compounds of the invention. Specifically, position13 must be modified to contain a π-bond in positions α, β to the ring.Preferred methods for preparing the synthetic polyketide thioesters areset forth in copending U.S. application Ser. No. 09/492,733 filed onJan. 27, 2000, which in turn claims priority from U.S. application Ser.No. 60/117,384 filed Jan. 27, 1999, which are incorporated herein byreference.

[0042] Recombinant forms of the 6-dEB PKS containing inactivatedketosynthase (KS) domains in the first module (KS 1) and appropriateorganisms modified to contain an expression system for this PKS aredescribed in PCT applications WO 97/02358, published Jan. 28, 1997 andWO 99/03986, published Jan. 28, 1999, incorporated herein by reference.

[0043] The polyketide resulting from expression of the modified PKS isthen isolated and purified, if desired, from the recombinantly modifiedorganism and fed to Saccharopolyspora erythraea, as shown in the firststep in FIG. 2, which contains the functionality for postpolyketidemodifications, including glycosylation. Other modifications includehydroxylation at positions 6 and 12. The resulting modified erythromycinis then isolated and chemically modified to obtain the compounds of theinvention. Synthetic methods for providing these modifications aredescribed in WO 98/09978 and U.S. Pat. No. 5,750,510, referencedhereinabove.

[0044] The general methods for synthesizing intermediates to compoundsof the invention are shown in FIGS. 2, 3, 6, 7, 9, 12 and 13.

[0045] The methods for synthesizing, from intermediates, the compoundsof the invention are shown in FIGS. 4, 5, 8, and 10.

[0046] The methods for modifying the substituents on the compounds ofthe invention or intermediates thereof are shown in FIGS. 9, 11 and 14.

[0047] The resulting anti-infective compound is active in vitro and invivo for activity against a panel of representative microorganisms. Thecompounds of the invention thus exhibit a sufficient diversity inspecificity to cover the spectrum of antibiotic activities desired.

[0048] For use in treating infectious disease, the compounds of theinvention are formulated into suitable compositions which will includetypical excipients, pharmaceutically acceptable counterions if thecompound is a salt, further additives as desired, such as antioxidants,buffers, and the like, and administered to animals or humans. The typesof formulations that are appropriate for these compounds are similar tothose for the macrolide antibiotics in general. Formulations may befound, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co., latest edition. The compounds can be administered by anydesired route, including injection, oral administration, transdermaladministration, transmucosal administration, or any combination. Thecompounds of the invention can also be administered with additionalactive ingredients if desired.

[0049] The compounds of the invention are of formulas(1)-(3), (1′)-(3′)or (101)-(103) as set forth above, as well as any stereoisomeric formsof these compounds as shown. The particular stereoisomers depicted arethose resulting from the preferred method of synthesis set forth aboveand exemplified herein; however, by modifying the expression system forthe PKS, or by altering the chirality of the diketide, or by syntheticchemical conversion, other stereoisomers may also be prepared.Additional chiral centers may be present in the substituents, such asR_(a), R_(d) and A, B, D and E, when any of A, B, D and E is not H. Thestereoisomers may be administered as mixtures, or individualstereoisomers may be separated and utilized as is known in the art.

[0050] The properties of the compounds of formulas (1)-(3) or (1′)-(3′)are defined by the substituents R_(a)-R_(e), A, B, D and E. Theproperties of the compounds of formulas (101)-(103) are defined by thesubstituents R_(a)-R_(e), L, T, Y and Z. Preferred embodiments of thesesubstituents are set forth hereinbelow. They contain moieties which aredefined as follows:

[0051] “Halogen” includes fluoro, chloro, bromo and iodo and mostpreferably, fluoro.

[0052] “Alkyl” refers to a saturated straight-chain, branched chain orcyclic hydrocarbyl moiety containing a specified number of carbons andthat may contain one or more suitable heteroatoms; similarly, alkenyland alkynyl refer to straight or branched chain or cyclic hydrocarbonsubstituents containing one or more double bonds or one or more triplebonds, respectively and containing one or more suitable heteroatoms.

[0053] “Aryl” refers to an aromatic substituent that may contain one ormore suitable heteroatoms such as phenyl, naphthyl, quinolyl, orphenanthryl.

[0054] “Arylalkyl”, “arylalkenyl” or “arylalkynyl” refers tosubstituents wherein an aryl group is linked to the substituted moietythrough an alkyl, alkenyl or alkynyl linkage respectively. Again, thenumber of carbons in the arylalkyl, arylalkenyl or arylalkynyl groupswill be specified.

[0055] “Amidoarylalkyl,” “amidoarylalkenyl,” or “amidoarylalkynyl” referto substituents wherein an aryl group is linked to the substitutedmoiety through an amido and an alkyl, alkenyl or alkynyl linkage,respectively. Again, the number of carbons in the amidoarylalkyl,amidoarylalkenyl or amidoarylalkynyl groups will be specified.

[0056] Thus, included among the defined substituents herein are“heteroalkyl,” “heteroalkenyl,” “heteroalkynyl,” “heteroaryl,”“heteroarylalkyl,” and the like. Suitable heteroatoms include N, O, andS.

[0057] All of the foregoing substituents may be unsubstituted or may befurther substituted. Typical substituents include R, —OR, —SR, —NR₂,—COR, —COOR, —CONR₂, —OOCR, —NRCOR, —OCONR₂, —CN, —CF₃, —NO₂, —SOR,—SO₂R, halogen wherein each R is independently H or is alkyl, alkenyl,alkynyl, aryl, arylalkyl, or the hetero forms of these as defined above.In addition, alkyl, alkenyl and alkynyl may be substituted by aryl orheteroaryl, which may, themselves, be further substituted.

[0058] “A derivatized oxime” is of the formula ═N—O—R, wherein R isother than H and is otherwise defined as above.

[0059] A “protecting group” for a hydroxy includes acyl groups, silylgroups, and the like. Suitable protecting groups are described byGreene, T. W., et al., in Protecting Groups in Organic Synthesis, 2^(nd)Ed., John Wiley & Sons, Inc. (1991), incorporated herein by reference.

[0060] The invention includes more preferred embodiments of the compounddefined above. R_(d) for compounds (1)-(3) or (1′)-(3′) is preferablybutyl, pentyl, methoxyethoxymethyl, isobutyl, methylcyclohexyl, phenyl,benzyl, ethylphenyl, 3-(benzyloxy)propyl, 2-(pyrimidin-2-ylthio)ethyl,propyl, fluoroethyl, chloroethyl, vinyl, 3-butenyl, or azidoethyl andmore preferably propyl, fluoroethyl, chloroethyl, vinyl, 3-butenyl, orazidoethyl. U.S. Ser. No. 60/117,384 filed Jan. 27, 1999 and U.S. Ser.No. 09/492,733 filed Jan. 27, 2000 both of which are incorporated hereinby reference describe various oligoketide thioesters, preferablydiketide thioesters, that can be incorporated at the C-13 position. Suchdiketide thioesters as described therein are incorporated into thecompounds of the invention and thus determine preferred R_(d) groups atthe C-13 position. Similarly, R_(x)—CH═C(R_(d)′) groups at the C-13position for compounds (101)-(103) are similarly described. Preferably,G is vinyl for intermediates for compounds (101)-(103).

[0061] In another preferred embodiment, R_(a) is H or lower C1-C3 alkyl,and more preferably methyl. R_(a) is also preferably arylalkenyl orarylalkynyl such as 3-arylprop-2-enyl or 3-arylprop-2-ynyl. Preferablythe aryl group in the preferred arylalkenyl or arylalkynyl embodimentsare 3-quinolyl, 4-quinolyl, 5-quinolyl, phenyl, 4-fluorophenyl,4-chlorophenyl, 4-methoxyphenyl, 6-quinolyl, 6-quinoxalyl,6-amino-3-quinolyl, or 4-isoquinolyl.

[0062] Synthesis of the Invention Compounds

[0063] As described above, the antibiotic starting materials for anyfurther chemical synthesis are prepared, preferably, by feeding asuitable diketide to a microorganism modified to contain an expressionsystem for the 6-dEB PKS containing a KS1 knockout, or by a host cellthat provides a methyl group at the 13 position followed by feeding theresulting polyketide to a recombinant strain of Saccharopolysporaerythraea that has been altered to eliminate production of 6-dEB. Astrain can be prepared that is able to hydroxylate both the 6- and12-positions, as shown schematically in FIG. 1, using the eryF and eryKhydroxylase genes or the 12-position only using the eryK hydroxylasegene and disrupting the eryF hydroxylase gene. In the latter case,—OR_(a) is replaced by —H. The recombinant S. erythraea strain, K40-67,is obtained by transforming an S. erythraea strain that produces highlevels of erythromycin A shown in FIG. 1 with a plasmid comprising amutated eryA1 sequence encoding an inactivated KS1 domain. By homologousrecombination, the resulting transformants now are unable to produce6-dEB as a competitor to the fed polyketide and, instead, hydroxylatethe 6-position and 12-position and glycosylate the 3-position and5-position of the modified polyketide that has been made in Streptomycesor other polyketide-producing transformant. If a macrolide having onlythe 12-position, and not the 6-position hydroxylated is desired (OR_(a)is replaced by H), an S. erythraea strain is constructed by disruptingthe eryF hydroxylase gene in strain K40-67.

[0064] Formation of the compounds of formulas (1)-(3), (1′)-(3′) and(101)-(103) requires the production of the erythronolide having ahydroxyl at the 12-position. The starting material may include any ofthe compounds (4)-(6) made by the method of FIG. 2:

[0065] The G substituent at position C-13 of intermediates (4)-(6) willbe chosen based on the final compound. G is R_(d) for intermediatecompounds of compounds (1)-(3) of the invention. In intermediatecompounds for compounds (101)-(103) of the invention, the position 13substituent in these intermediates must contain a point of unsaturationand thus must be capable of forming the compounds of the invention.Therefore, G is R_(x)—CH═C(R_(d)′)— for intermediates of compounds(101)-(103) of the invention. R_(x) can be H or R_(d)′, and preferablyalkyl, aryl or arylalkyl, and more preferably H, so that the substituentis a reactive vinyl group. R_(x) is lost during ozonolysis in theintermediate formation of the compounds of the invention.

[0066] The glycosylation reactions for the production of theerythromycins result in the diglycosylated forms analogous to naturallyoccurring erythromycins. If the compounds of formula (5) or (6) are tobe prepared from the initial diglycosylated product, the hydroxyl groupof the cladinose ring (attached to position 3) may then need to beprotected for subsequent modification of the macrolide substituents. Thecladinose essentially serves as a 3-OH protecting group and can beremoved after compound (3), (3′) or (103) is formed. FIG. 7, which willbe described in further detail below, illustrates the synthesis schemewherein the cladinose moiety is first removed, leaving a 3-hydroxy groupthat is subsequently protected so that alkylation of the 6-hydroxy ispossible. FIG. 3 illustrates the retention of the cladinose moietiesafter the position 6 alkylation is completed.

[0067] The modified erythromycins of the invention, in addition tomodification at C-13, contain an —OH group at position 6 unless OR_(a)is replaced by H as described above. In the embodiment of compounds(101)-(103) having a 12-13 fused ring, the hydroxyl group at position 6can be converted before or after the carbamate or carbonate ring isformed, as shown in FIGS. 3 and 7 (before) and FIG. 11 (after). Toconstruct the compounds of formulas (1), (2), (3), (1′), (2′), (3′),(101), (102), and (103) where position 6 OR_(a), the compound of formula(I) (see FIG. 2) is provided with protecting groups which form oneembodiment of R_(c) and R_(e). Such protection is effected usingsuitable protecting reagents such as acetic anhydride, benzoicanhydride, benzochloro formate, hexamethyldisilazane, or a trialkylsilylchloride in an aprotic solvent. Aprotic solvents include, for example,dichloromethane, chloroform, tetrahydrofuran, N-methyl pyrrolidone,dimethyl sulfoxide (DMSO), dimethyl formamide (DMF) and the like.Mixtures may also be used. Protection of both sugar hydroxyls in formula(I) may be done simultaneously or sequentially.

[0068] In addition to protecting the 2′ and 4″ hydroxyl groups of thetwo glycose residues, the keto group at position 9 of the macrolide ringmust also be protected. Typically, this is effected by converting theketo group to a derivatized oxime.

[0069] Particularly preferred embodiments for R in the formula ═NORinclude unsubstituted or substituted alkyl (1-12C), substituted orunsubstituted aryl (6-10C), alkyl (1-12C), substituted or unsubstitutedheteroaryl (6-10C), alkyl (1-12C), and heteroalkyl (such as substituentsof the formula CR′₂OR′ wherein each R′, in addition to beingindependently embodied as R as set forth above, may, together with theother, form a cycloalkyl ring (3-12C)). A preferred derivatized oxime isof the formula ═NOR wherein R is isopropoxycyclohexyl.

[0070] With the 9-keto group and the 2′ and 4″ hydroxyls protected, itis then possible to alkylate the 6-hydroxy group in the compound offormula (I) by reaction with an alkylating agent in the presence ofbase. Alkylating agents include alkyl halides and sulfonates. Forexample, the alkylating agents may include methyl tosylate,2-fluoroethyl bromide, cinnamyl bromide, crotonyl bromide, allylbromide, propargyl bromide, and the like. The alkylation is conducted inthe presence of base, such as potassium hydroxide, sodium hydride,potassium isopropoxide, potassium t-butoxide, and an aprotic solvent.

[0071] The choice of alkylating agent will depend on the nature of thesubstituents R_(a) to be included. As set forth above, R_(a) can besubstituted or unsubstituted alkyl (1-10C), substituted or unsubstitutedalkenyl (2-10C), substituted or unsubstituted alkynyl (2-10C)substituted or unsubstituted aryl (3-20C) or substituted orunsubstituted arylalkyl (4-20C). Particularly preferred areunsubstituted alkyl, alkenyl, or alkynyl, or substituted forms of thesewherein the substituents include one or more halogen, hydroxy, alkoxy(1-6C), oxo, SO₂R (1-6C), N₃, CN, and NR₂ wherein R is H, substituted orunsubstituted alkyl (including cycloalkyl) (1-12C), substituted orunsubstituted alkenyl (including cycloalkenyl) (2-12C), alkynyl(including cycloalkynyl) (2-12C), substituted or unsubstituted aryl(6-10C), including the hetero forms of the above.

[0072] Especially preferred are methyl, allyl and ethyl.

[0073] Protection of the groups described above are also described inU.S. application Ser. Nos. 09/551,162 and 09/550,045 which areincorporated herewith in their entirety.

[0074] Once the alkylation of the 6-hydroxyl is completed, the sugarresidues and the macrolide ring may be deprotected. Deprotection of theglycoside moieties is conducted as described by Green, T. W., et al., inProtective Groups in Organic Synthesis, infra. Similar conditions resultin converting the derivatized oxime to ═NOH. If formation of theunderivatized oxime is not concurrent with deprotection, the conversionto the oxime is conducted separately.

[0075] The oxime can then be removed and converted to a keto group bystandard methods known in the art. Deoximating agents include inorganicsulfur oxide compounds such as sodium hydrogen sulfite, sodiumpyrosulfate, sodium thiosulfate, and the like. In this case, proticsolvents are used, such as water, methanol, ethanol, isopropanol,trimethyl silanol and mixtures of these. In general, the deoximationreaction is conducted in the presence of an organic acid.

[0076] At this point in the process, or later, after the compound offormula (4) has been converted to the compounds of formulas (5) or (6)or to any of compounds (1)-(3), (1′)-(3′), or (101)-(103), as furtherdescribed below, the group introduced at the 6-hydroxyl can further bemanipulated. Conveniently, the initial substitution may provide a6-O-allyl, i.e., O—CH₂CH═CH₂, which can further be derivatized byreduction to give the 6-O propyl compound, or be treated with osmiumtetroxide to provide the 2,3-dihydroxypropyl compound, which can furtherbe esterified at each oxygen atom. The O-allyl derivative can also beoxidized with m-chloroperoxybenzoic acid in an aprotic solvent toprovide the epoxy compound which can be opened with amines orN-containing heteroaryl compounds to provide compounds with N-containingside-chains, or can be oxidized under Wacker conditions to provide thesubstituent O—CH₂—C(O)—CH₃, or can be ozonized to provide the aldehyde.The aldehyde can then be converted to the oxime or reacted with asuitable amine and reduced in the presence of a borohydride reducingagent to provide an amine. The oxime can also be converted to a nitrileby reaction with a dehydration agent in an aprotic solvent. The O-allylderivative can also be reacted with an aryl halide under Heck conditions(Pd(II) or Pd(O), phosphine and amine or inorganic base) to provide a3-aryl prop-2-enyl derivative. This derivative can then be reduced withhydrogen and palladium on carbon to provide a 3-arylpropyl derivative.If the initial substituent R_(a) is a 2-propyne, similar reactions canbe employed to provide alterations in the side-chain, includingarylation.

[0077] In order to convert the compound of formula (4) into the compoundof formula (6) by first removing the cladinose moiety, the compound offormula (4) is treated with mild aqueous acid or with a deglycosylatingenzyme. Suitable acids include hydrochloric, sulfuric, chloroacetic,trifluoroacetic and the like, in the presence of alcohol. Reaction timesare typically 0.5-24 hours at a temperature of −10-35° C. During thisreaction, the 2′ group of the remaining sugar is protected as set forthabove and deprotected subsequent to the decladinizing reaction. Theresulting hydroxyl group at the 3-position of the macrolide ring is thenoxidized to the ketone using a modified Swem oxidation procedure. Inthis procedure, an oxidizing agent such as N-chlorosuccinimide-dimethylsulfide or a carbodiamide-dimethylsulfoxide is used. Typically, acompound of formula (4) is added to pre-formed N-chlorosuccinimide anddimethyl sulfide complex in a chlorinated solvent such as methylenechloride at −10-25° C. After being stirred for 0.5-4 hours, a tertiaryamine such as triethylamine is added to produce the corresponding ketoneand the 2′ protecting group is then removed.

[0078] In order to halogenate the macrolide at position 2 (convertingR_(b)═H to halogen), the compound of formula (6), where R_(b)═H, istreated with a base and an electrophilic halogenating reagent such aspyridinium perbromide or N-fluorobenzene sulfonic acid. The position 2can be halogenated at any time after the 3 keto group is prepared.

[0079] In one embodiment of compounds (1)-(3) and (1′)-(3′), where C13is not part of a fused ring, the appropriate substituent such as vinyl,ethenyl, butenyl or azido at the C-13 position can be furthermanipulated. For example, an amidoacetate salt of the compound of theinvention can be derivatized using an arylacetyl chloride to yield anarylamino alkyl group on the C-13 position. Preferably the C13derivatives of an azido group take place before the ketolide is formed.Derivations of an ethenyl group can take place either before or afterthe ketolide is formed.

[0080] In order to obtain the compounds of formula (5), the compoundresulting from the deglycosylation reaction of formula (4) is treatedwith a dehydrating agent such as carbonyl diimidazole and case.

[0081] In embodiments where the C9 is not part of a fused ring, in orderto prepare compounds of formulas (101)-(103) wherein one of Z and Y is Hand the other OH or protected OH or is an amino derivative as describedabove, either the carbonyl or oxime or derivatized oxime is reducedusing a suitable reducing agent, such as sodium borohydride, Raneynickel/H₂ or reductive amination with the use of sodium cyanoborohydrideand an amine. Substituted amines can also be obtained by alkylation.

[0082] I. Cyclization of Fused Rings at Positions 10-11, and 9 and 11

[0083] Intermediates (7)-(9) can then be prepared from intermediates(4)-(6).

[0084] It will be noted that the presence of the 12-hydroxyl group isrequired. The hydroxyl groups of the sugar moieties are protected asdescribed above and the resulting protected compounds are then reactedwith sodium hexamethyldisilazide and carbonyldiimidazole which resultsin dehydration to obtain a π-bond at position 10-11 and derivatizationof the 12-hydroxyl to provide functionality in the macrolide ring asshown in compounds (7)-(9). FIG. 4 illustrates the reaction sequencefrom compound (6) to compound (7) in the first step.

[0085] Reaction of compounds (7)-(9) with a diamine having the formula

[0086] provides the intermediate compounds of formulas (10)-(12). Oneexample is shown in FIG. 4 for compounds (7) and (10) in the secondstep.

[0087] These intermediates are compounds of the formulas (10)-(12):

[0088] where A, B, D and E are described above.

[0089] Cyclizing compounds (10)-(12) with dilute mineral or organicacid, optionally deprotecting provides compounds (1)-(3) and (1′)-(3′)of the invention, as illustrated in FIG. 4 for intermediate compounds(10) and compounds (1) and (1′) of the invention, which compounds can befurther isolated.

[0090] In accordance with FIG. 4, compound (6) is converted to compound(7) by reaction with carbonyldiimidazole and an alkali metal hydridebase, such as sodium hydride, lithium hydride or potassium hydride in asuitable aprotic solvent at from about 0° C. to ambient temperature.Compound (7) may also be prepared by reaction of the diol compound (6),or cyclic carbonate I₇, prepared as described in FIG. 6, by reactionwith carbonyldiimidazole and sodium or lithium hydride under similarconditions. Compound (7) is then reacted with diamine

[0091] having substituents A, B, D and E as defined above, as shown inFIG. 4, in a suitable solvent such as aqueous acetonitrile, DMF oraqueous DMF, to give the bicyclic compound (10). Compound (10) is thencyclized by treatment with dilute acid, such as acetic acid or HCl in asuitable organic solvent such as ethanol or propanol and deprotected asdescribed above to give the tricyclic ketolide (1). Alternatively, the2′-protecting group of the bicyclic ketolid (10) may be removed prior tocyclization using the methods described in FIG. 2. Compounds of formula(1) may be reduced to compounds of formula (1′) by treatment with areducing agent selected from hydrogen in the presence of palladiumcatalyst, alkyl borohydride and lithium aluminum hydride in a suitableorganic solvent.

[0092] Alternatively, reaction of compounds (7)-(9) with an amine havingthe formula

[0093] wherein A, B, D and E are as defined above, and Y is hydroxy,provide the compounds of formulas (10′)-(12′):

[0094] Treating compounds (10′)-(12′) with triphenylphosphine anddiphenylphosphoryl azide and diethylazodicarboxylate in tetrahydrofarangives the analogous compound wherein Y is N₃, and removing thedeprotecting group gives the analogous compound wherein Y is N₃ andR_(c) is H; and then further treating the resulting compounds with areducing agent selected from the group consisting oftriphenylophosphine-water, hydrogen with a catalyst, sodium borohydride,and dialkylaluminum hydride, yields compounds (10′)-(12′) wherein R_(c)is H.

[0095] Cyclizing compounds (10′)-(12′) with a dilute mineral or organicacid yields compounds (1′)-(3′) of the invention, which can be furtherisolated.

[0096] More specifically, FIG. 5 illustrates an alternative preparationof compounds of formula (1). Starting material (7) is reacted with abeta-aminoalcohol

[0097] (Y═OH) in a suitable solvent system such as aqueous acetonitrile,DMF or aqueous DMF at 0-70° C. to give (10′) which is converted to theazide with a Mitsunobu reaction using triphenylophosphine anddiphenylphosphoryl azide and DEAD in tetrahydrofuran. Alternatively, thehydroxy group in (10′) may be activated by treatment with sulfonylchloride, alkyl or aryl sulfonic anhydride or trifluoromethanesulfonicanhydride in an aprotic solvent. The activated hydroxy group is thenconverted to the corresponding azide by reaction with lithium azide orsodium azide in an aprotic solvent. The 2′-protecting group is thenremoved as described above, and the azide is reduced to the amine (10′).Suitable reducing reagents are triphenylphosphine-water, hydrogen with acatalyst, sodium borohydride, or dialkylaluminum hydride in theappropriate solvent for these reactions, as is well known in the art.Compound (10′) is then cyclized as described in FIG. 5 above.

[0098] Of course, if the substrate for the ring formation is a compoundof formula (4), a compound of the formula (3′) results; modificationscan then be conducted to convert the compound of formula (3′) tocompounds of formulas (1′) and (2′), as described above. Under thesecircumstances, the keto group would be protected by a derivatized oximeand the 2′ hydroxyl group would be protected by a protecting group. Suchmodifications include removal of the cladinose moiety by acidhydrolysis; oxidizing the 3-hydroxyl group; and deprotecting theprotected hydroxyl and keto groups.

[0099] According to the alternate procedure shown in FIG. 7a, theintermediate compound (I₁), which is the 9-oxime compound oferythromycin A, is subjected to acid hydrolysis with dilute mineral ororganic acid as described previously to remove the cladinose moiety andgive intermediate compound (I₂). The oxime compound (I₂) is thenconverted to the protected oxime compound (I₃) wherein V is ═N—O—R¹where R¹ is a protecting group, by reaction with the appropriatelysubstituted oxime protecting reagent. The 3 and 2′-hydroxy groups of(I₃) are then protected, preferably with a trimethylsilyl protectinggroup, to give compound (I₄). Compound (I₄) is then alkylated asdescribed previously to give compound I₅), and compound (I₅) is firstdeoximated as described above then the deoximated product is convertedto the compound (I₆). FIG. 7b shows compound (I₆) is then deprotectedand oxidized to the 3-ketolide derivative, intermediate compound (10),by procedures described previously. Intermediate compound I₆ can also bedeprotected and dehydrated to form intermediate compound (11), alsoshown in FIG. 7b.

[0100] The cyclic carbonate compounds of the formulas 17 and 18 shown inFIG. 6 are prepared from compounds (4)-(6) using the procedure describedby Baker et al., J Org Chem (1988) 53:2340 which is incorporated hereinby reference. The 2′ or 2′,4″-protected compounds of formulas (4)-(6)are first converted to the cyclic carbonates by reaction withcarbonyldiimidazole and sodium hexamethyldisilazide. FIG. 6 illustratesthe conversion of the compound having formula (6) to the compound havingthe formula I₈.

[0101] As mentioned earlier, the 6-position substituent can bemanipulated after the compounds (1)-(3) are formed. The O-allylderivative can be reacted with an aryl halide under Heck conditions(Pd(II) or Pd(O), phosphine and amine or inorganic base). In addition,for example, compound (10) can be prepared wherein R_(a) is—CH₂—CH—N—OR_(h) and R_(h) is H or C₁-C₃-alkyl, aryl substitutedC₁-C₃-alkyl, or heteroaryl substituted C₁-C₃-alkyl. In this method, afirst compound (10), wherein R_(a) is —CH₂—CH═CH₂, is treated with ozoneto form a second compound (10) wherein R_(a) is —CH₂—CH═O. Then compound(10) wherein R_(a) is —CH₂—CH═O is further treated with a hydroxylaminecompound having the formula NH₂—O—R_(h), wherein R_(h) is as previouslydefined; and optionally deprotected, and the desired compound may beisolated.

[0102] In another embodiment of the invention is a process for preparinga compound (10) wherein R_(a) is —CH₂—CH₂—NH—R_(i) where R_(i), with theatom to which it is attached, form a 3-10 membered substituted orunsubstituted heterocycloalkyl ring. The method comprises reductivelyaminating compound (10) wherein R_(a) is —CH₂—CH═O with an aminecompound having the formula —NH₂—R_(i), wherein R_(i) is as previouslydefined; and optionally deprotecting, and isolating the desiredcompound.

[0103] Novel methods of synthesis of the compounds of the invention arealso provided.

[0104] Exemplary Embodiments

[0105] The compounds of formulas (1), (2), (3), (1′), (2′) and (3′) aredefined by their various substituents. Table 1 illustrates compoundswithin the scope of the present invention which are:

[0106] of formula (1) or (1′) wherein R_(b) is H, Cl, F or Br, and R_(c)is H;

[0107] of formula (2) or (2′) wherein R_(c) is H; and

[0108] of formula (3) or (3′) wherein R_(b) is H, Cl, F or Br, R_(c) isH, and R_(e) is H. TABLE 1 R_(d) R_(a) —CH₃ —CH₂CH₂-Φ —CH═CH₂—CH₂CH═CH-Φ —CH₂CH₂CH₃ —CH₂CH₂NHCH₃ —CH₃ —CH₂CHOHCH₃ —CH(CH₃)₂ —CH₂-Φ—CH₃ —CH₂—CH═CH₂ —CH₃ —CH₂—CH═CH-(3-quinolyl) —CH₃—CH₂—CH₂—CH₂-(3-quinolyl) —CH₃ —CH₂—CH═CH-(2-methyl-6-quinolyl) —CH₃—CH₂—CH═CH-(5-isoquinolyl) —CH₃ —CH₂—CH═CH-(3-bromo-6-quinolyl) —CH₃—CH₂—C═CH-(6-methoxy-2-naphthyl) —CH₃ —CH₂—C≡C-(2-phenylethenyl) —CH₃—CH₂—C≡C-(3-quinolyl) —CH₃ —CH₂—C≡C-naphthyl —CH₃—CH₂—C≡C-(6-methyl-2-naphthyl) —CH₃ —CH₂—C≡C-(3-(2-furanyl)-6-quinolyl)—CH═CH₂ —CH₃ —CH₂OH —CH₂—C═CH-(4-fluorophenyl) —CH₂OH—CH₂—C═CH-(3-quinolyl) —CH₂OH —CH₂—C═CH-(6-quinolyl) —CH₂OCH₃—CH₂—C═CH-(3-pyridyl) —CH₂CH₂CH₃ —CH₂—C═CH-(3-quinolyl) —CH₂CH₂CH₃—CH₂—C═CH-(6-chloro-3-quinolyl) —CH₂CH₂CH₃ —CH₂—C═CH-(4-quinolyl)—CH₂CH₂CH₃ —CH₂—C═CH-(6-chloro-3-quinolyl) —CH₂CH₂CH₃—CH₂—C═CH-(6-hydroxy-3-quinolyl) —CH₂CH₂CH₃—CH₂—C═CH-(6-methoxy-3-quinolyl) —CH₂CH₂CH₃—CH₂—C═CH-(6-aminocarbonyl-3- quinolyl) —CH₂CH₂CH₃—CH₂—C═CH-(3-(2-thiophenyl)-6- quinolyl) —CH₂CH₂CH₃—CH₂—C═CH-(6-hydroxy-2-naphthyl) —CH₂CH₂CH₃ —CH₂—C≡C-(3-quinolyl)—CH₂CH₂CH₃ —CH₂—C≡(6-chloro-2-naphthyl) —CH₂CH₂CH₃ —CH₂—C≡C-(6-quinolyl)—CH₂CH₂CH₃ —CH₂CH₂NHCH₂CH₂-(2- chlorophenyl) —CH₃ —CH₂CH₂NH₂ —CH₃ OR_(a)replaced by H —CH₃ —CH₃ —CH₃ OR_(a) replaced by H —CH₃ ″ —CH₃ ″ —CH₃—CH₂CHClCH₃ —CH₃ ″ —CH₃ ″ —CH₃ —CH₃ —CH₂CH₂CH₃ OR_(a) replaced by H—CH₂CH₂CH₃ ″ —CH₂CH₂CH₃ —CHCH(OCH₃)CH₃ —CH₂CH₂CH₃ —CH₃ —CH₂CH₂CH₃—CH₂CH₂CH₃ —CH₂CH₂CH₃ —CH₂CHBrCH₃ —CH₃ —CH₂CHOHCH₃ —CH₂CH₂CH₃ —CH₂CH₂CH₃—CH₃ —CH₂CH═CH₂ —CH₃ —CH₂CH═CH-(3-quinolyl) —CH₃ —CH₂CH═CH₂ —CH₃—CH₂CH═CH-(3-quinolyl) —CH₃ —CH₂CH═CH₂ —CH₃ —CH₂CH═CH-(3-quinolyl) —CH₃—CH₂CH═CH₂ —CH₃ —CH₂CH═CH-(3-quinolyl) —CH₃ —CH₂CH₂CH_(2-(3-quinolyl))—CH₃ —CH₂CH═CH₂ —CH₃ —CH₂CH═CH-(3-quinolyl) —CH₃ —CH₂CH₂CH₂-(3-quinolyl)

[0109] II. Cyclization of Fused Ring at Position 12-13

[0110] Intermediates (107)-(109) or (107′)-(109′) can then be preparedfrom intermediates (4)-(6).

[0111] It will be noted that the presence of the 12-hydroxyl group isrequired. As mentioned above the 6-OH can be converted to —OR_(a) beforeor after the carbamate or carbonate ring is formed. FIG. 8 illustratesthe reaction sequence from a compound analogous to compound (4) to acompound analogous to compounds (109) and (109′) in the first steps,where the 6-OH is not yet converted to —OR_(a).

[0112] To prepare compounds (107)-(109), the vinyl or other group havinga π-bond in positions α, β to the ring at the 13 position of any ofcompounds (4)-(6) is first converted to an aldehyde by ozonolysis,preferably by using ozone and dimethyl sulfide and triphenyl phosphitein methanol.

[0113] The aldehyde at position 13 is then converted to an imine andthen an amine using compounds of the formulas H₂NR, H₂NOR, H₂NNHCOR,H₂NN═CHR or H₂NNHR, where R is H or R_(a), and NaBH₃CN. R_(x) is lostduring ozonolysis in the formation of the aldehyde. The amine is thenconverted to compounds (107)-(109) using PhOCOCl. Reaction of compounds(107)-(109) with sodium hydride in THF provides a compound analogous tothe compounds of formulas (110)-(112) below, which are also analogous tocompounds of formulas (101)-(103) wherein L is carbonyl and T is NR, asshown in FIG. 8 for the 6-hydroxy analogs to compounds (109) and (103)in the second step.

[0114] To prepare compounds (107′)-(109′), the aldehyde is prepared fromthe vinyl as described above and then NaBH₄ is used to form the alcoholin compounds (107′)-(109′).

[0115] Reaction of compounds (107′)-(109′) with PhOCOCl and then sodiumhydride and finally treatment with methanol provides the 6-hydroxyanalogs of the cyclic carbonate compounds of formulas (110′)-(112′)below, which are compounds of formulas (101)-(103) wherein L is carbonyland T is —O—, as shown in FIG. 8 for the 6-hydroxy analogs of compounds(109′) and (103) in the second step.

[0116] The 6-hydroxy group can then be converted to R_(a) and theprotected 2′ and 4″-hydroxy groups can be deprotected as describedabove.

[0117] The compounds of the invention of the formulas (110)-(112) or(110′)-(112′) are formed from the intermediate compounds (107)-(109) or(107′)-(109′) respectively as discussed above:

[0118] where R_(j) represents the substituents on the nitrogen asdescribed above. The preparation of compounds of the formulas(110)-(112) or (110′)-(112′) follows the procedure described by Baker etal. J Org Chem (1988) 53:2340, which is incorporated herein byreference.

[0119] Alternate or additional procedures may be used to preparecompounds (110)-(112) where R_(j) is not H.

[0120] For example, the compounds of formulas (110)-(112) wherein R_(j)is H can be reacted with an alkylating agent which is of the formulaR-halogen to replace the hydrogen on the ring nitrogen with an alkylgroup, as shown in FIG. 9.

[0121] Further, compounds (110)-(112) that do not contain an acyl groupas a substituent on the nitrogen of T can be formed by treatment of suchcompounds (110)-(112) with an acylating agent selected from the groupconsisting of R(CO)-halogen or (RCO)₂O to give compounds (107)-(109)wherein T is —N— and R_(j) is —NH—COR.

[0122] Treatment of compounds (110)-(112) where R_(j) is —NH₂ with analdehyde R—CHO, wherein R is as defined previously gives compounds(110)-(112) wherein R_(j) is —N═CHR.

[0123] Treatment of compounds (110)-(112), where R_(j) is —NH₂ with analkylating agent having the formula R-halogen, wherein R is as definedpreviously, gives the compounds (110)-(112) where R_(j) is R.

[0124] Of course, if the substrate for the ring formation is a compoundof formula (4) wherein G is R_(x)—CH═C(R_(d)′)—, a compound of theformula (103) results; modifications can then be conducted to convertthe compound of formula (103) to compounds of formulas (101) and (102),as described above. Under these circumstances, the keto group would beprotected by a derivatized oxime and the 2′ hydroxyl group would beprotected with a protecting group. Such modifications include removal ofthe cladinose moiety by acid hydrolysis; oxidizing the 3-hydroxyl group;and deprotecting the protected hydroxyl and keto groups.

[0125] According to the alternate procedure shown in FIG. 10, the 6hydroxy group is converted to R_(a) before the cyclic carbamate orcarbonate is formed, as in the process of FIG. 2. This scheme followsScheme 7a for the synthesis of compounds I₁-I₅. Specifically, theintermediate compound (I₁), which is the 9-oxime compound oferythromycin A, is subjected to acid hydrolysis with dilute mineral ororganic acid as described previously to remove the cladinose moiety andgive intermediate compound (I₂). The oxime compound (I₂) is thenconverted to the protected oxime compound (I₃) wherein V is ═N—O—R¹where R¹ is a protecting group, by reaction with the appropriatelysubstituted oxime protecting reagent. The 3 and 2′-hydroxy groups of(I₃) are then protected, preferably with a trimethylsilyl protectinggroup, to give compound (L). Compound (I₄) is then alkylated asdescribed previously to give compound (I₅), and compound (I₅) is firstdeoximated as described above then the deoximated product is convertedto the compound (I₁₀₆) by the procedures described for preparation ofthe 6-hydroxy analog of compound (103) from the 6-hydroxy analog ofcompound (4) in FIG. 8. FIG. 10 shows compound (I₁₀₆) is thendeprotected and oxidized to the 3-ketolide derivative, compound (110) ofthe invention, wherein L is CO and T is —NR_(j) or —O— by proceduresdescribed previously. Intermediate compound (I₁₀₆) can also bedeprotected and dehydrated to form compound (111) of the invention, alsoshown in FIG. 10.

[0126] As mentioned earlier, the 6-position substituent can bemanipulated after the compounds (101)-(103) are formed. For example,compound (110) can be prepared wherein R_(a) is —CH₂—CH—N—OR_(h) andR_(h) is H or C₁-C₃-alkyl, aryl substituted C₁-C₃-alkyl, or heteroarylsubstituted C₁-C₃-alkyl. In this method, a first compound (110), whereinR_(a) is —CH₂—CH═CH₂, is treated with ozone to form a second compound(110) wherein R_(a) is —CH₂—CH═O. Then, compound (110), wherein R_(a) is—CH₂—CH═O, is further treated with a hydroxylamine compound having theformula NH₂—O—R_(h), wherein R_(h) is as previously defined; and isoptionally deprotected, and the desired compound may be isolated. In apreferred embodiment of the process immediately above, R is H.

[0127] In another embodiment of the invention is a process for preparinga compound (110) wherein R_(a) is —CH₂—CH₂—NH—R_(i) where R_(i), withthe atom to which it is attached, form a 3-10 membered substituted orunsubstituted heterocycloalkyl ring. The method comprises reductivelyaminating compound (110) wherein R_(a) is —CH₂—CH═O with an aminecompound having the formula —NH₂—R_(i), wherein R_(i) is as previouslydefined; and optionally deprotecting, and isolating the desiredcompound.

[0128] In addition, manipulation to the hydroxyl group at the 6 positioncan be conducted after the carbamate or carbonate rings are formed, asshown in FIG. 11a. In FIG. 11a the 6-hydroxy analog of the compound offormulas (112) or (112′), are converted to compound (103), and further,compound (101) of the invention.

[0129] According to this alternative procedure illustrated in FIG. 11a,the keto group at the 9 position in compound (I_(a)) is converted to anoxime as in compound (I_(b)) and then to a derivatized oxime in compound(I_(c)), which will form the protecting group as described above.

[0130] The hydroxyl groups at the 2′ and 4″ positions of the cladinosemoieties are then provided with trimethylsilyl protecting groups asshown in compound (I_(d)). The protection of the 2′ and 4″ hydroxyls andthe keto group at position 9, prepares the compound for alkylation ofthe hydroxyl group at the 6 position as described above, which gives thecompound of formula (103).

[0131] The 9-keto and 2′ and 4″ hydroxy groups then can be deprotectedto restore the 2′ and 4″ hydroxy groups and an ═NOH group at the 9position which can be further converted to the original 9-keto group asdescribed above.

[0132]FIG. 11b illustrates the removal of the cladinose moiety at the 3position to convert the compound of formula (103) to compound (I_(e)).The 2′-hydroxy group must be protected to give the compound (I_(f)) sothat the 3-hydroxy can be oxidized to a 3-carbonyl to give compound(101). Further manipulation of the —OR_(a) group takes place to form avariation of compound (101) before final deprotection to provide acompound of formula (101) having a hydroxyl group at the 2′ position ofthe cladinose.

[0133] In order to prepare compounds of formulas (4)-(6) or (101)-(103)wherein one of Z and Y is H and the other OH or protected OH or is anamino derivative as described above, either the carbonyl or oxime orderivatized oxime is reduced using a suitable reducing agent.Substituted amines are obtained by alkylation.

[0134] Novel methods of synthesis of the compounds of the invention arealso provided.

[0135] Exemplary Embodiments

[0136] The compounds of formulas (101), (102) and (103) are defined bytheir various substituents. Table 2 illustrates compounds within thescope of the present invention which are:

[0137] of formula (101) wherein R_(b) is H, Cl, F, or Br, L is CO, andR_(c) is H;

[0138] of formula (102) wherein R_(c) is H and L is CO; and

[0139] of formula (103) wherein R_(b) is H, Cl, F, or Br, R_(c) is H, Lis CO, and R_(e) is H. TABLE 2 R_(d)′ R_(a) Y Z T —CH₃ —CH₂CH₂-φ ═O —NH——CH═CH₂ —CH₂CH═CH-φ ═O —N(CH₃)— —CH₂CH₂CH₃ —CH₂CH₂NHCH₃ ═NOH —N(NHCH₃)——CH₃ —CH₂CHOHCH₃ ═NOCH₂CH₃ —N(OCH₃)— —CH(CH₃)₂ —CH₂-φ H OH —N(N═CH₂)——CH₃ —CH₂—CH═CH₂ ═O —O— or —NH— —CH₃ —CH₂—CH═CH-(3-quinolyl) ═O —O— or—NH— —CH₃ —CH₂—CH₂—CH₂-(3-quinolyl) ═O —O— or —NH— —CH₃—CH₂—CH═CH-(2-methyl-6-quinolyl) ═O —O— or —NH— —CH₃—CH₂—CH═CH-(5-isoquinolyl) ═O —O— or —NH— —CH₃—CH₂—CH═CH-(3-bromo-6-quinolyl) ═O —O— or —NH— —CH₂—CH₂—C═CH-(6-methoxy-2-naphthyl) ═O —O— or —NH— —CH₂—CH₂—C≡C-(2-phenylethenyl) ═O —O— or —NH— —CH₂ —CH₂—C≡C-(3-quinolyl) ═O—O— or —NH— —CH₂ —CH₂—C≡C-naphthyl ═O —O— or —NH— —CH₂—CH₂—C≡C-(6-methyl-2-naphthyl) ═O —O— or —NH— —CH₂—CH₂—C≡C-(3-(2-furanyl)-6-quinolyl) ═O —O— or —NH— —CH═CH₂ —CH₃ ═O —O—or —NH— —CH₂OH —CH₂—C═CH-(4-fluorophenyl) ═O —O— or —NH— —CH₂OH—CH₂—C═CH-(3-quinolyl) ═O —O— or —NH— —CH₂OH —CH₂—C═CH-(6-quinolyl) ═O—O— or —NH— —CH₂OCH₃ —CH₂—C═CH-(3-pyridyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(3-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(6-chloro-3-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(4-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(6-hydroxy-3-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(6-methoxy-3-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(6-aminocarbonyl-3-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(3-(2-thiophenyl)-6-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C═CH-(6-hydroxy-2-naphthyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C≡C-(3-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C≡C-(6-chloro-2-naphthyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂—C≡C-(6-quinolyl) ═O —O— or —NH— —CH₂CH₂CH₃—CH₂CH₂NHCH₂CH₂-(2-chlorophenyl) ═O —O— or —NH— —CH₃ —CH₂CH₂NH₂ ═O —O—or —NH— —CH₃ OR_(a) replaced by H —NH₂ H —O— or —NH— —CH₃ —CH₃ —NH₂ H—O— or —NH— —CH₃ OR_(a) replaced by H

H —O— or —NH— —CH₃ ″

H —O— or —NH— —CH₃ ″

H —O— or —NH— —CH₃ —CH₂CHClCH₃ H

—O— or —NH— —CH₃ ″ H

—O— or —NH— —CH₃ ″ H

—O— or —NH— —CH₃ —CH₃

H —O— or —NH— —CH₂CH₂CH₃ OR_(a) replaced by H H

—O— or —NH— —CH₂CH₂CH₃ ″ —NH₂ H —O— or —NH— —CH₂CH₂CH₃ —CHCH(OCH₃)CH₃

H —O— or —NH— —CH₂CH₂CH₃ —CH₃ H

—O— or —NH— —CH₂CH₂CH₃ —CH₂CH₂CH₃

H —O— or —NH— —CH₂CH₂CH₃ —CH₂CHBrCH₃ H

—O— or —NH— —CH₃ —CH₂CHOHCH₃ ═NOCHCH₃ —O— or —NH— —CH₂CH₂CH₃ —CH₂CH₂CH₃—NH₂ H —O— or —NH— —CH₃ —CH₂CH═CH₂ ═O —N(CH₃) —CH₃—CH₂CH═CH-(3-quinolyl) ═O —N(CH₃) —CH₃ —CH₂CH═CH₂ ═O N(CH₂CH₂N(CH₃)₂)—CH₃ —CH₂CH═CH-(3-quinolyl) ═O N(CH₂CH₂N(CH₃)₂) —CH₃ —CH₂CH═CH₂ ═ON(CH₂CH═CH₂) —CH₃ —CH₂CH═CH-(3-quinolyl) ═O N(CH₂CH═C-(3-quinolyl)) —CH₃—CH₂CH═CH₂ ═O N(NH₂) —CH₃ —CH₂CH═CH-(3-quinolyl) ═O N(NH₂) —CH₃—CH₂CH₂CH₂-(3-quinolyl) ═O N(NH₂) —CH₃ —CH₂CH═CH₂ ═O N(NH₂) —CH₃—CH₂CH═CH-(3-quinolyl) ═O N(NH₂) —CH₃ —CH₂CH₂CH₂-(3-quinolyl) ═O N(NH₂)H —CH₂CH₂-φ ═O —NH— H —CH₂CH═CH-φ ═O —N(CH₃)— H —CH₂CH₂NHCH₃ ═NOH—N(NHCH₃)— H —CH₂CHOHCH₃ ═NOCH₂CH₃ —N(OCH₃)— H —CH₂-φ H OH —N(N═CH₃)— H—CH₂—CH═CH₂ ═O —O— or —NH— H —CH₂—CH═CH-(3-quinolyl) ═O —O— or —NH— H—CH₂—CH₂—CH₂-(3-quinolyl) ═O —O— or —NH— H—CH₂—CH═CH-(2-methyl-6-quinolyl) ═O —O— or —NH— H—CH₂—CH═CH-(5-isoquinolyl) ═O —O— or —NH— H—CH₂—CH═CH-(3-bromo-6-quinolyl) ═O —O— or —NH— H—CH₂—C═CH-(6-methoxy-2-naphthyl) ═O —O— or —NH— H—CH₂—C≡C-(2-phenylethenyl) ═O —O— or —NH— H —CH₂—C≡C-(3-quinolyl) ═O —O—or —NH— H —CH₂—C≡C-naphthyl ═O —O— or —NH— H—CH₂—C≡C-(6-methyl-2-naphthyl) ═O —O— or —NH— H—CH₂—C≡C-(3-(2-furanyl)-6-quinolyl) ═O —O— or —NH— H —CH₃ ═O —O— or —NH—H —CH₂—C═CH-(4-fluorophenyl) ═O —O— or —NH— H —CH₂—C═CH-(6-quinolyl) ═O—O— or —NH— H —CH₂—C═CH-(3-pyridyl) ═O —O— or —NH— H—CH₂—C═CH-(6-chloro-3-quinolyl) ═O —O— or —NH— H —CH₂—C═CH-(4-quinolyl)═O —O— or —NH— H —CH₂—C═CH-(6-hydroxy-3-quinolyl) ═O —O— or —NH— H—CH₂—C═CH-(6-methoxy-3-quinolyl) ═O —O— or —NH— H—CH₂—C═CH-(6-aminocarbonyl-3-quinolyl) ═O —O— or —NH— H—CH₂—C═CH-(3-(2-thiophenyl)-6-quinolyl) ═O —O— or —NH— H—CH₂—C═CH-(6-hydroxy-2-naphthyl) ═O —O— or —NH— H—CH₂—C≡C-(6-chloro-2-naphthyl) ═O —O— or —NH— H —CH₂—C≡C-(6-quinolyl) ═O—O— or —NH— H —CH₂CH₂NHCH₂CH₂-(2-chlorophenyl) ═O —O— or —NH— H—CH₂CH₂NH₂ ═O —O— or —NH— H OR_(a) replaced by H —NH₂ H —O— or —NH— H—CH₃ —NH₂ H —O— or —NH— H OR_(a) replaced by H

H —O— or —NH— H ″

H —O— or —NH— H ″

H —O— or —NH— H —CH₂CHClCH₃ H

—O— or —NH— H ″ H

—O— or —NH— H ″ H

—O— or —NH— H —CH₃

H —O— or —NH— H OR_(a) replaced by H H

—O— or —NH— H ″ —NH₂ H —O— or —NH— H —CHCH(OCH₃)CH₃

H —O— or —NH— H —CH₃ H

—O— or —NH— H —CH₂CH₂CH₃

H —O— or —NH— H —CH₂CHBrCH₃ H

—O— or —NH— H —CH₂CH₂CH₃ —NH₂ H —O— or —NH— H —CH₂CH═CH₂ ═O —N(CH₃) H—CH₂CH═CH-(3-quinolyl) ═O —N(CH₃) H —CH₂CH═CH₂ ═O N(CH₂CH₂N(CH₃)₂) H—CH₂CH═CH-(3-quinolyl) ═O N(CH₂CH₂N(CH₃)₂) H —CH₂CH═CH₂ ═O N(CH₂CH═CH₂)H —CH₂CH═CH-(3-quinolyl) ═O N(CH₂CH═C-(3-quinolyl)) H —CH₂CH═CH₂ ═ON(NH₂)

EXAMPLES

[0140] The following examples are intended to illustrate but not tolimit the invention.

[0141] Compound numbers and designations are found in the IllustrativeSchemes.

[0142] In these examples, in the first general step of the method, a6-deoxyerythronolide B (6-dEB) derivative compound is prepared byfermentation of a recombinant Streptomyces host cell.

[0143] The fermentation to produce 15-methyl-6-deoxyerythronolide, anintermediate compound for the compounds (1)-(3) or (1′)-(3′) and14,15-dehydro-6-deoxyerythronolide B, an intermediate for the compounds(1)-(3), (1′)-(3′) and (101)-(103), requires a synthetic diketideintermediate to be fed to the fermenting cells. The preparation of thesesynthetic diketides is described in Example 1. These synthetic diketidesare substrates for a 6-deoxyerythronolide B synthase (DEBS) that isunable to act on its natural substrate (propionyl CoA) due to a mutationin the ketosynthase domain of module 1 of DEBS. This recombinant DEBS isprovided by plasmid pJRJ2 in Streptomyces coelicolor CH999. S.coelicolor CH999 is described in U.S. Pat. No. 5,672,491, incorporatedherein by reference. A derivative of S. coelicolor CH999, S. coelicolorK39-02, that has been genetically modified to include a ptpA gene, isdescribed in U.S. patent application Ser. No. 09/181,833, incorporatedherein by reference can also be employed for this purpose.

[0144] Plasmid pJRJ2 encodes the eryAI, eryAII, and eryAIII genes; theeryAI gene contained in the plasmid contains the KS1 null mutation. TheKS1 null mutation prevents formation of the 6-deoxyerythronolide Bproduced by the wild-type gene unless exogenous substrate is provided.Plasmid pJRJ2 and a process for using the plasmid to prepare novel13-substituted erythromycins are described in PCT publication Nos.99/03986 and 97/02358 and in U.S. patent application Ser. Nos.08/675,817, filed Jul. 5, 1996; 08/896,323, filed Jul. 17, 1997; and09/311,756, filed May 14, 1999, each of which is incorporated herein byreference. The exogenous substrates provided can be prepared by themethods and include the compounds described in PCT patent applicationNo. PCT/US00/02397 and U.S. patent application Ser. No. 09/492,733, bothfiled Jan. 27, 2000, by inventors G. Ashley et al., and both of whichclaim priority to U.S. patent application Ser. No. 60/117,384, filedJan. 27, 1999, each of which is incorporated herein by reference. PKSgenes other than the ery genes can also be employed; suitable genesinclude the KS1 null mutation containing oleandolide and megalomicin PKSgenes described in U.S. patent application Ser. Nos. 60/158,305, filedOct. 8, 1999 and 09/428,517, filed Oct. 28, 1999, and PCT applicationNo. US99/24478, filed Oct. 22, 1999, each of which is incorporatedherein by reference.

[0145] For compounds (1)-(3) or (1′)-(3′), where there is no fused ringat C13, the fermentation to produce the intermediate14-nor-6-deoxyerythronolide B does not require diketide feeding, becausethe desired compound is produced by the recombinant host cellStreptomyces coelicolor CH999/pCK7. Plasmid pCK7 is described in U.S.Pat. No. 5,672,491 and comprises the DEBS genes. A derivative of plasmidpCK7, pKOS011-26, can also be used. The host cell comprising pKOS011-26and a recombinant ptpA gene is S. coelicolor 27-26/pKOS011-26. Thesehost cells produce both 6-deoxyerythronolide B and14-nor-6-deoxyerythronolide, due to the incorporation of propionyl CoAand acetyl CoA, both of which serve as substrates for DEBS.

[0146] The fermentation of Streptomyces coelicolor CH999/pJRJ2 and S.coelicolor CH999/pCK7 is described in Example 2. The isolation of the6-deoxyerythronolide products resulting from this fermentation can beachieved by separation.

[0147] The isolated products are then added to the fermentation broth ofSaccharopolyspora erythraea strains to make other useful intermediatecompounds of the invention. The S. erythraea strains catalyze thebiosynthesis and attachment of sugar residues to the 3 and 5 positionsof the 6-dEB derivative compounds. These strains also comprise afunctional eryK gene product and so hydroxylate the 6-dEB derivativecompounds at the 12 position. The strains differ in regard to whether afunctional eryF gene product is produced. If so, then the compoundsproduced are hydroxylated at the 6 position as well. If not, then a6-deoxyerythromycin A derivative is produced. These S. erythraeafermentations are described in Example 3, together with the isolation ofthe erythromycin A derivative compounds from the fermentation broth.

[0148] The isolated products are then used as intermediates in thechemical synthesis of other intermediate compounds of the invention. Forerythromycin A derivative intermediates that comprise a 6-hydroxyl,Examples 4-6 describe the process for alkylating the compounds to makethe 6-O-alkyl intermediates of the invention. The schematic for thesereactions is shown in FIG. 3.

[0149] Examples 7-9 describe the conversion of the above-describedcompounds of formula (4) to compounds of formula (6), and correspondingcompounds that are the 10,11-anhydro forms. This is shown schematicallyin FIG. 12.

[0150] Examples 10-12 also set forth the process for making the10,11-anhydro compounds of formula (4) or (6), but wherein OR_(a) isreplaced by H. The reaction scheme for these conversions is shown inFIG. 13.

[0151] Examples 13 and 14 describe the synthesis of intermediatecompound (5) by cleavage of the cladinose moiety intermediate compound(4). Example 15 describes the protection of the C9 carbonyl of compound(5).

[0152] Examples 16-23 set forth the synthesis of compounds (1)-(3) ofthe invention using various intermediate compounds and various diaminederivatives.

[0153] Examples 24-26 set forth in the synthesis of compounds(101)-(103) of the invention using various intermediates.

[0154] Examples 27-28 describe R_(a) and R_(c) conversions of compoundsof the invention.

[0155] Examples 29 illustrates the halogenation of the 2-position.

[0156] Example 30 illustrates the conversion of 15-azidoerythromycin Ainto 15-amidoerythromycins, as shown in FIG. 14.

Example 1 Preparation of Diketide Thioesters

[0157] The processes used to prepare the N-acetylcysteaminethioesters(NAcS) used to feed the recombinant Streptomyces host cells to make the14,15-dehydro-6-deoxyerythronolide B intermediate compounds aredescribed in this Example. The synthesis protocols described below arealso described in U.S. provisional patent application Ser. No.60/117,384, filed Jan. 27, 1999, U.S. utility patent application Nos.08/846,247 filed Apr. 30, 1997, 09/073,538 filed May 6, 1998, and U.S.utility patent application Ser. No. 09/492,733, filed Jan. 27, 2000,which are incorporated herein by reference in their entirety.

[0158] In similar fashion, (2S,3R)-2-methyl-3-hydroxy-4-pentenoate-NAcS(Preparation G), which is used to prepare the14,15-dehydro-6-deoxyerythronolide B intermediate, is prepared fromreacting(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinone(Preparation F) with N-acetylcysteamine (Preparation B).(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinone(Preparation F) is prepared from(4S)-N-Propionyl-4-benzyl-2-oxazolidinone (Propionyl-NOx; PreparationC).

[0159] A. N,S-diacetylcysteamine

[0160] Cysteamine hydrochloride (50.0 g) is added to a 1 L 3-neck roundbottom flask fitted with a magnetic stir bar, 2 addition funnels, and apH electrode. Water (300 mL) is added, and the stirred solution iscooled on ice. The pH is adjusted to 8.0 by addition of 8 N KOH. Aceticanhydride (125 mL) is placed in one addition funnel, and 8N KOH (350 mL)is placed in the other addition funnel. The acetic anhydride is addeddropwise to the cysteamine solution, with 8 N KOH being added so as tokeep the reaction pH at 8+/−1. After addition of acetic anhydride iscomplete, the pH was adjusted to 7.0 using 1 N HCl and the mixture isallowed to stir for 75 min. on ice. Solid NaCl is added to saturation,and the solution is extracted 4 times using 400 mL portions of CH₂Cl₂.The organic extracts are combined, dried over MgSO₄, filtered, andconcentrated under reduced pressure to yield 68.9 g (97% yield) of apale yellow oil, which crystallizes upon standing at 4° C.

[0161] B. N-acetylcysteamine

[0162] N,S-diacetylcysteamine (42.64 g) is placed in a 2 L round bottomflask fitted with a magnetic stirrer, and dissolved in 1400 mL of water.The flask is purged with N₂, and the mixture is chilled in an ice bath.Potassium hydroxide (49.42 g) is added, and the mixture is stirred for 2hr. on ice under inert atmosphere. The pH is adjusted to 7 using 6 NHCl, and solid NaCl is added to saturation. The mixture is extracted 7times with 500 mL portions of CH₂Cl₂. The organic extracts are combined,dried over MgSO₄, filtered, and concentrated under reduced pressure toyield 30.2 g (96% yield) of product. This material is distilledimmediately prior to use, bp 138-140° C./7 mmHg.

[0163] C. (4S)-N-propionyl-4-benzyl-2-oxazolidinone (Propionyl-NOx)

[0164] A dry, 1 L three-necked round bottomed flask equipped with a 500mL addition funnel and a stir bar was charged with 20 g of(4S)-4-benzyl-2-oxazolidinone, capped with septa and flushed withnitrogen. Anhydrous THF (300 mL) was added by cannula and the resultingsolution was cooled with a −78° C. bath of dry ice/isopropanol. Theaddition funnel was charged with 78 mL of n-butyllithium (1.6 M inhexane) by cannula, which was added in a slow stream to the reaction.Distilled propionyl chloride (bp 77-79° C.), 8.0 mL, was added rapidlyvia syringe. The reaction was allowed to stir for 30 min. in the dryice/isopropanol bath.

[0165] The reaction was removed from the cold bath, allowed to warmto >0° C., and quenched with 50 mL of saturated aqueous NH₄Cl. Themixture was concentrated to a slurry on a rotary evaporator. The slurrywas extracted three times with 250 mL portions of ethyl ether. Theorganic extracts were combined and washed with 50 mL each of saturatedaqueous NaHCO₃ and brine, dried with MgSO₄, filtered, and concentratedto give a yellow oil. The material crystallized upon sitting. Thecrystals were triturated once with cold (−20° C.) hexanes to give 21.0 g(80% yield) of white crystalline material, m.p. 41-43° C.

[0166] APCI-MS: m/z=234 (MH+), 178, 117. 1H-NMR (360 MHz, CDCl₃):δ7.2-7.4 (5H,m); 4.67 (1H,m,H4); 4.14-4.22 (2H,m,H5); 3.30 (1H,dd,J=3,13Hz,benzylic); 2.89-3.03 (2H,m,H2′); 2.77 (1H,dd,J=9,13,benzylic); 1.20(3H,t,J=7 Hz,H2′).

[0167] D.(4S)-N-[(2S,3R)-2-methyl-3-hydroxyhexanoyl]-4-benzyl-2-oxazolidinone

[0168] A dry, 2 L three-necked round bottomed flask equipped with a 500mL addition funnel, a low-temperature thermometer, and a stir bar wascharged with 19.84 g of N-propionyl-oxazolidinone, capped with septa andflushed with nitrogen. Anhydrous dichloromethane (100 mL) was added bycannula, and the resulting solution was cooled to −65° C. in a bath ofdry ice/isopropanol. The addition funnel was charged by cannula with 100mL of dibutylboron triflate (1.0 M in dichloromethane), which was addedin a slow stream to the reaction. Triethylamine (15.6 mL) was addeddropwise by syringe, keeping the reaction temperature below −10° C. Thereaction was then transferred to an ice bath and allowed to stir at 0°C. for 30 min. After that period, the reaction was placed back into thedry ice/isopropanol bath and allowed to cool to −65° C. Butyraldehyde(8.6 mL) was added rapidly by syringe, and the reaction was allowed tostir for 30 min.

[0169] The reaction was transferred to an ice bath and the additionfunnel was charged with 100 mL of a 1 M aqueous phosphate solution, pH7.0 (the phosphate solution is comprised of equal molar amounts of mono-and dibasic potassium phosphate). The phosphate solution was added asquickly as possible while keeping the reaction temperature below 10° C.The addition funnel was then charged with 300 mL methanol which wasadded as quickly as possible while keeping the reaction temperaturebelow 10° C. Finally, the addition funnel was charged with 300 mL of 2:1methanol:30% hydrogen peroxide. This was added dropwise to ensure thatthe temperature was kept below 10° C. The reaction was stirred for onehr. after completion of addition. The solvent was then removed on arotary evaporator until a slurry remained. The slurry was extracted 4times with 500 mL portions of ethyl ether. The combined organic extractswere washed with 250 mL each of saturated aqueous sodium bicarbonate andbrine. The extract was then dried with MgSO₄, filtered, and concentratedto give a slightly yellow oil. The material was then chromatographed onSiO₂ using 2:1 hexanes:ethyl acetate (product Rf=0.4) resulting in 22.0g (85% yield) of title compound as a colorless oil.

[0170] APCI-MS: m/z 306 (MH+); 1H-NMR (360 MHz, CDCl₃): δ7.2-7.4 (5H,m,phenyl); 4.71 (1H,m,H4); 4.17-4.25 (2H,m,H5); 3.96 (1H,m,H3′); 3.77(1H,dq,J=2.5,7 Hz, H2′); 3.26 (1H,dd,J=4,13 Hz,benzylic); 2.79(H,dd,J=9,13 Hz,benzylic); 1.5-1.6 (2H,m,H4′); 1.3-1.5 (2H,m,H5′); 1.27(3H,d,J=7 Hz,2′-Me); 0.94 (3H,t,J=7 Hz,H6′).

[0171] E. (2S,3R)-2-methyl-3-hydroxyhexanoate N-acetylcysteaminethioester N-acetylcysteamine was distilled at 130° C./7 mm Hg to give acolorless liquid at room temperature. A dry, 1 L three-necked roundbottomed flask equipped with a 500 mL addition funnel and a stir bar wascapped with septa and flushed with nitrogen. The flask was then chargedwith 10.7 mL of N-acetylcysteamine by syringe and with 400 mL ofanhydrous THF by cannula. The mixture was cooled with a MeOH/ice bath.Butyllithium (64 mL of 1.6 M in hexanes) was added dropwise by syringe,resulting in formation of a white precipitate. After stirring for 30min., trimethylaluminum (51 mL of 2.0 M in hexanes) was added dropwiseby syringe. The reaction became clear after addition oftrimethylaluminum and was allowed to stir an additional 30 min. Duringthis period, 20.5 g (0.068 mol) of(4S)-N-[(2S,3R)-2-methyl-3-hydroxylhexanoyl]-4-benzyl-2-oxazolidinonewas put under a blanket of nitrogen and dissolved in 100 mL of anhydrousTHF; this solution was then transferred in a slow stream by cannula intothe reaction. The resulting reaction mixture turned a yellow-green colorand was allowed to stir for 1 hr. The reaction was finished when thestarting material could no longer be seen by thin-layer chromatographicanalysis (ca. 1 hr.).

[0172] The reaction was treated with enough saturated oxalic acid togive a neutral reaction with pH paper (approximately 90 mL). Thesolvents were then removed on a rotary evaporator to give a whiteslurry. The slurry was extracted six times with 250 mL portions of ethylether. The organic extracts were combined and washed with brine, driedwith MgSO₄, filtered, and concentrated to give a slightly yellow oil.The thioester product was purified by flash chromatography on SiO₂ using1:1 hexanes:EtOAc until the elution of 4-benzyl-2-oxazolidinone. At thatpoint, the solvent system was switched to 100% EtOAc to give purefractions of diketide thioester. The product fractions were combined andconcentrated to give 14.9 g (89% yield) of title compound. This compoundis referred to as the propyl diketide thioester in Example 2.

[0173] APCI-MS: m/z 248 (MH+); 1H-NMR (360 MHz, CDCl₃): δ5.8 (br s,1H);3.94 (dt,1H), 3.46 (m,2H), 3.03 (dt,2H), 2.71 (dq,1H), 1.97 (s,3H), 1.50(m,2H), 1.37 (m,2H), 1.21 (d,3H), 0.94 (t,3H).

[0174] F.(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinone

[0175] A dry, 2 L three-necked round bottomed flask equipped with a 500mL addition funnel, a low-temperature thermometer, and a stir bar wascharged with 20.0 g of propionyl oxazolidinone A, capped with septa andflushed with nitrogen. Anhydrous dichloromethane (100 ml) was added andthe resulting solution was cooled to −15° C. in a bath of methanol/ice.Dibutylboron triflate (100 mL of 1.0 M in dichloromethane) was added ina slow stream via the addition funnel at such a rate as to keep thereaction temperature below 3° C. Diisopropylethylamine (17.9 mL) wasadded dropwise by syringe, again keeping the internal temperature below3° C. The reaction was then cooled to −65° C. using a dryice/isopropanol bath. Acrolein was added over 5 min. by syringe. Thereaction was allowed to stir for 30 min. after completion of addition.

[0176] The reaction was then transferred to an ice bath and the additionfunnel was charged with 120 mL (0.1 mol) of a 1 M aqueous phosphatesolution, pH 7.0 (the phosphate solution is comprised of equal molaramounts of mono- and dibasic phosphate). The phosphate solution wasadded as quickly as possible while keeping the reaction temperaturebelow 10° C. The addition funnel was then charged with 400 mL ofmethanol that were added as quickly as possible while keeping thereaction temperature below 10° C. Finally, the addition funnel wascharged with 400 mL of 2:1 methanol:30% hydrogen peroxide by initialdropwise addition to keep the temperature below 10° C. The reaction wasstirred for one hour. The solvent was removed using a rotary evaporator,leaving a slurry. The slurry was extracted 4 times with 500 mL portionsof ethyl ether. The organic extracts were combined and washed with 250mL each of saturated sodium bicarbonate and brine, then dried withMgSO₄, filtered, and concentrated to give a slightly yellow oil.Trituration with hexane induced crystallization. Recrystallization fromether by addition of hexane resulted in 13.67 g (55% yield) of product.

[0177] 1H-NMR (360 MHz, CDCl₃): δ7.2-7.4 (m,5H); 5.86 (ddd,1H), 5.35(dt,1H), 5.22 (dt,1H), 4.71 (m,1H), 4.51 (m,1H), 4.21 (m,2H), 3.89(dq,1H), 3.26 (dd,1H), 2.80 (dd,1H), 1.25 (d,3H).

[0178] G. (2S,3R)-2-methyl-3-hydroxy-4-pentenoate N-acetylcysteaminethioester

[0179] N-acetylcysteamine was distilled at 130° C./7 mm Hg to give acolorless liquid at room temperature. A dry, 1 L three-necked roundbottomed flask equipped with a 500 mL addition funnel and a stir bar wascapped with septa and flushed with nitrogen. The flask was then chargedwith 7.5 mL of N-acetylcysteamine by syringe and with 500 mL ofanhydrous THF by cannula. The reaction was then cooled with a MeOH/icebath. Butyllithium (44 mL of 1.6 M in hexane) was added dropwise bysyringe. A white precipitate formed as the n-BuLi was added. Afterstirring for 30 min., 35.5 mL (0.071 mol) of trimethylaluminum (2.0 M inhexane) were added dropwise by syringe. The reaction became clear afteraddition of trimethylaluminum and was allowed to stir an additional 30min.(4S)-N-[(2S,3R)-2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinonefrom Preparation F (13.6 g) was put under a blanket of nitrogen,dissolved in 50 mL of anhydrous THF, and this solution was thentransferred in a slow stream by cannula into the reaction. The resultingreaction mixture turned a yellow-green color and was allowed to stir for1 hr. The reaction was judged to be finished when starting materialcould no longer be seen by thin-layer chromatography (ca. 30 min.).

[0180] Enough saturated oxalic acid was added to give a neutral reactionwith pH paper (approximately 60 mL). The solvents were then removed byrotary evaporator to give a white slurry. The slurry was extracted sixtimes with 250 mL portions of ethyl ether. The organic extracts werecombined, washed with brine, dried with MgSO₄, filtered, andconcentrated to give a slightly yellow oil. The thioester was thenpurified by flash chromatography on SiO₂. The column was run with 1:1hexanes:ethyl acetate until the elution of oxazolidinone. At that point,the eluent was switched to 100% ethyl acetate to give pure fractions ofproduct. The fractions were combined and concentrated to give 7.7 g (71%yield) of title compound product. This product is referred to as thevinyl diketide thioester in Example 2.

[0181] 1H-NMR (360 MHz, CDCl₃): δ5.82 (ddd,1H), 5.78 (br s, 1H), 5.32(dt,1H), 5.21 (dt,1H), 4.47 (m,1H), 3.45 (m,2H), 3.04 (m,2H), 2.81(dq,1H), 1.96 (s,3H), 1.22 (d,3H).

Example 2

[0182] Preparation of Erythronolide

[0183] A. 15-methyl-6-deoxyerythronolide B (Compound P, R_(a)=H,G=Rd₄=propyl)

[0184]Streptomyces coelicolor CH999/pJRJ2 is described in U.S. patentapplication Ser. Nos. 08/896,323, filed Jul. 17, 1997, and 08/675,817,filed Jul. 5, 1996, each of which is incorporated herein by reference.Plasmid pJRJ2 encodes a mutated form of DEBS in which the ketosynthasedomain of module 1 (KS1) has been inactivated via mutagenesis (KS1°). S.coelicolor strains comprising this plasmid that are fed (2S,3R)-2-methyl-3-hydroxyhexanoate-N-acetylcysteamine (Preparation E,propyl diketide) of Example 1 produce 15-methyl-6-deoxyerythronolide B.

[0185] A 1 mL vial of the CH999/pJRJ2 working cell bank is thawed andthe contents of the vial are added to 50 mL of Inoculum Medium 1 in a250 mL baffled flask. The flask is placed in an incubator/shakermaintained at 30±1° C. and 175±25 RPM for 48±10 hours. The 50 mL cultureis then added to a 2.8 L baffled flask containing 500 mL of InoculumMedium 1. This flask is incubated in an incubator/shaker at 30±1° C. and175±25 RPM for 48±10 hours. The 500 mL culture is divided equally amongten 2.8 L baffled flasks each containing 500 mL of Inoculum Medium 1.All flasks are then incubated as described previously.

[0186] A 150 L fermenter is prepared by sterilizing 100 L of ProductionMedium 1 at 121° C. for 45 minutes. After incubation, all 10 flasks arecombined in a 5 L sterile inoculation bottle and aseptically added to a150 L fermenter. The fermenter is controlled at 30° C., pH 6.5 byaddition of 2.5 N H₂SO₄ and 2.5 N NaOH, dissolved oxygen ≧80% airsaturation by agitation rate (500-700 RPM), air flow rate (10-50 LPM),and/or back pressure control (0.1-0.4 bar). Foam is controlled by theintermittent addition of a 50% solution of Antifoam B.

[0187] At 24±5 hours (2S,3R)-2-methyl-3-hydroxyhexanoyl-N-acetylcysteamine (propyl diketide,Preparation E in Example 1) is added to a final concentration of 1 g/L.Propyl diketide is prepared by solubilizing in dimethyl sulfoxide at aratio of 1:4 (diketide to DMSO) and then filter sterilized (0.2 μm,nylon filter). Production of 15-methyl-6-deoxyerythronolide B(15-methyl-6dEB) ceases on day 7 and the fermenter is harvested. Thefermentation broth is centrifuged at 20,500 g in an Alpha Laval AS-26centrifuge. The product is predominantly in the centrate; thecentrifuged cell mass is discarded.

[0188] This process has also been completed in a 1000 L fermenter (700 Lworking volume). The inoculum process is identical to the above processexcept that the 150 L fermenter is charged with Inoculum Medium 1 andthe 1000 L fermenter is charged with Production Medium 1. The fermenteris controlled at 30° C., pH 6.5 by addition of 2.5-5 N H₂SO₄ and 2.5-5 NNaOH, dissolved oxygen ≧70% air saturation by agitation rate (140-205RPM), air flow rate (100-200 LPM), and/or back pressure control (0.2-0.5bar). Foam is controlled by the addition of a 50% solution of Antifoam Bas needed. At 24±5 hours racemic2-methyl-3-hydroxyhexanoyl-N-propionylcysteamine (300 grams) is added tothe 1000 L fermenter. The fermenter is harvested at 4.6 days bycentrifugation as described above.

[0189] Media used in this process include the following: Inoculum Medium1 Component Concentration KNO₃ 2 g/L Yeast extract 20 g/L Hycase SF 20g/L FeSO₄-7H₂O 25 mg/L NaCl (12.5% stock) 4 mL/L MgSO₄ (12.5% stock) 4mL/L MnSO₄-H₂O (0.5% stock) 1 mL/L ZnSO₄-7H₂O (1.0% stock) 1 mL/LCaCl₂-2H₂O (2.0% stock) 1 mL/L

[0190] Post-sterile Additions:

[0191] 1) 1 mL/L of 50 mg/ml Thiostrepton in 100% DMSO, sterilefiltered.

[0192] 2) 1 mL/L 100% Antifoam B silicon emulsion (J. T. Baker),autoclaved.

[0193] 3) 40 mL of 500 g/L glucose, sterile filtered. Production Medium1 Component g/L Corn Starch 45 Corn steep liquor 10 Dried, inactivatedbrewers yeast 10 CaCO₃ 1

[0194] Post-sterile Additions for Production Medium 1

[0195] 1) 1 mL/L of 50 mg/ml Thiostrepton in 100% DMSO, sterilefiltered.

[0196] 2) 1 mL/L of 100% Antifoam B (J. T. Baker), autoclaved.

[0197] After centrifugation, the centrate is filtered. The filtrate(approximately 700 L) are passed through an Amicon Moduline column(20×350 cm) containing 20 L of HP20 resin (Mitsubishi). The flow rateduring loading is 4 L/minute with a pressure drop below 8 psi. Afterloading the resin is washed with 20 L of water and then 40 L of 30%methanol. 15-methyl-6dEB is eluted using 100% methanol. Four 12 Lfractions were collected with fractions 2, 3 and 4 containing all of thedetectable 15-methyl-6dEB. The 15-methyl-6dEB product pool is dilutedwith 36.7 L of water giving 75 L of a clear solution. This solution isloaded directly onto a 5 L Amicon Vantage Column containing HP20SS resin(Mitsubishi). Column loading is carried out at 1 L/minute. The column iseluted with 20 L of 65% methanol, 20 L of 70% methanol, 20 L of 80%methanol, and finally 20 L of 100% methanol. A total of 16×5 L fractionswere collected. The 80% fractions along with the last 70% fraction werecombined (25 L) and evaporated to dryness. The resulting residue isdissolved in 1 L of 100% methanol, filtered, evaporated, and dried in avacuum oven at 40° C. This process resulted in 33 g of a solid productcontaining 93% 15-methyl-6dEB.

[0198] B. 14,15-dehydro-6-deox erythronolide B (Compound P, R_(a)=H,G=R_(d)=allyl

[0199]S. coelicolor strains comprising this plasmid that are fed(2S,3R)-2-methyl-3-hydroxy-4-pentenoate NAc Cysteamine thioester(Preparation G) of Example 1 produce 14,15-dehydro-6-deoxyerythronolideB when prepared in accordance with the process described in PreparationA above to produce 15-methyl-6-deoxyerythronolide B.

[0200] C. 14-nor-6-deoxyerythronolide B (Compound P, R_(a)=H,G=Rd_(d)=methyl)

[0201] Similarly, 14-nor-6-deoxyerythronolide B is produced using S.coelicolor CH999/pCK7 host, without using a diketide thioester, whenprepared in accordance with the process described in Example 2A.

Example 3 Preparation of Erythromycins

[0202] The 6-dEB derivative compounds produced in Example 2 is convertedto erythromycin derivatives using a recombinant strain ofSaccharopolyspora erythraea. For production of erythromycins having boththe 6- and 12-hydroxyl groups, the S. erythraea strain used was K40-67or K39-14V. This strain was created by transforming an S. erythraeastrain capable of producing high levels of erythromycin A with apWHM3-derived plasmid comprising a mutated eryA1 sequence encoding aninactivated KS1 domain. By homologous recombination, the resultingtransformants were rendered incapable of producing 6-deoxyerythronolideB. Thus the dEB analog fed is not subject to competition forhydroxylation at the 6-position. For production of erythromycinderivatives having only the 12-hydroxyl group, the S. erythraea strainused was K39-07. This strain was constructed from strain K40-67 bydisruption of the eryF hydroxylase gene; this destroys the ability tohydroxylate the analog at the 6-position. Both strains were fermentedunder substantially similar conditions, as described below.15-methyl-erythromycin A: 15-methyl-erythromycin A is produced accordingto the following protocol: A 1 mL vial of the K39-14V working cell bankis thawed and the contents of the vial are added to 50 mL of InoculumMedium 2 in a 250 mL baffled flask. The flask is placed in anincubator/shaker maintained at 34±1° C. and 175±25 RPM for 48±10 hours.The 50 mL culture is then added to a 2.8 L baffled flask containing 500mL of Inoculum Medium 2. The flask is incubated in an incubator/shakerat 34±1° C. and 175±25 RPM for 48±10 hours. The 500 mL culture isdivided equally among ten 2.8 L baffled flasks each containing 500 mL ofInoculum Medium 2. All flasks are then incubated as describedpreviously.

[0203] A 150 L fermenter is prepared by sterilizing 100 L of ProductionMedium 2 at 121 ° C. for 45 minutes. After incubation, all 10 flasks arecombined in a 5 L sterile inoculation bottle and aseptically added to a150 L fermenter. The fermenter is controlled at 34° C., pH 7.0 byaddition of 2.5 N H₂SO₄ and 2.5 N NaOH, dissolved oxygen ≧80% airsaturation by agitation rate (500-700 RPM), air flow rate (15-50 LPM),and/or back pressure control (0.1-0.4 bar). Foam is controlled by theaddition of a 50% solution of Antifoam B.

[0204] At 24±5 hours a 58-60 mL/hour 15% dextrin (w/v) feed isinitiated. The dextrin solution is continuously mixed during the feedperiod. At 24±5 hours 25 grams of 15-methyl-6dEB (Preparation A inExample 2) are added to the fermenter. The 15-methyl-6dEB is prepared bysolubilizing 25 grams of 15-methyl-6dEB in 400-600 mL of 100% ethanoland filtering (0.2 μm, nylon filter). Conversion of 15-methyl-6dEB to15-methyl-erythromycin A ceases after 60±10 hours and the fermenter isharvested. The fermentation broth is centrifuged at 20,500 g in an AlphaLaval AS-26 centrifuge. The product is predominantly in the centrate;the centrifuged cell mass is discarded.

[0205] Media used in this process include the following: Inoculum Medium2 Component g/L Corn Starch 16.0 Corn dextrin 10.0 Soy Meal Flour 15.0CaCO₃ 4.0 Corn steep liquor 5.0 Soy Bean Oil 6.0 NaCl 2.5 (NH₄)₂SO₄ 1.0

[0206] Post-sterile Addition

[0207] 1 mL/L 100% Antifoam B (J. T. Baker), autoclaved. ProductionMedium 2 Component g/L Corn Starch 17.5 Corn Dextrin (Type 3) 16.0 SoyMeal Flour 16.5 CaCO₃ 4.0 Corn steep liquor 6.0 Soy Bean Oil 3.0 NaCl3.5 (NH₄)₂SO₄ 1.0

[0208] Centrifuged fermentation broth (127 L) containing 34 g of thetarget molecule is passed through 18.3 L of HP20 sorbent packed into anAmicon P350 Moduline 2 chromatography column. At 4 L/min loading,backpressure is found to be less than 5 psi. Following loading, theresin is washed with 20 L deionized water and then 40 L of 30% methanol.15-methyl-erythromycin A is eluted using 54 L of 100% methanol. Theproduct pool is evaporated using a Buchi rotary evaporator (R-152). Thesolids were dissolved in a minimal amount of 100% methanol, filtered andthe filtrate evaporated to dryness. This resulted in 123 g of materialcontaining 30% 15-methyl-erythromycin A by weight. 80 grams of the 30%material is extracted twice with 1 L of 40° C. acetone. The acetoneextract is filtered, and the filtrate is dried on the inside surface ofa 20 L rotary evaporation flask. The solids were extracted with 9:1hexane to acetone three times at 40° C. The organic extracts were pooledand evaporated to dryness giving 32 g of solids enriched (68%) in15-methyl-erythromycin A. The product pool from the acetone/hexaneextraction is dissolved in 1 L of methanol to which an equal amount ofwater is added. The methanol solution is loaded onto a HP20SSchromatography column (Kontes) previously washed and equilibrated with50% methanol. Column dimensions were 4.8×115 cm. Column loading withrespect to 15-methyl-erythromycin A is 11 g/L. The column is washed with50% (0.8 L) and 60% (8 L) methanol in water. Elution of the targetmolecule is carried out using 70% (8L), 80% (16 L) and 85% (8 L)methanol in water. 1 L fractions were collected. Fractions 11-29 werecombined, evaporated and dried in a vacuum oven giving 23 g of productwith 93% purity.

[0209] The following compounds are also produced by this methodology:14-norerythromycin A (G=R_(d)=Me); 14,15-dehydro-erythromycin A(G=R_(d)=allyl, R_(x)=R_(d)′=H); 14-nor-6-deoxy-erythromycin A;14,15-dehydro-6-deoxy-erythromycin A; and 15-methyl-6-deoxy-erythromycinA. When used to make 3-descladinose-3-oxo-derivatives, the erythromycinA derivatives were not separated from the erythromycin C derivatives;instead, mixtures of the erythromycin A and erythromycin C compoundswere used as starting materials for chemical derivatization.

[0210] These products were extracted and purified as follows:

[0211] In general, fermentation broths are brought to pH 8.0 by additionof NaOH and ethanol is added (0.1 L/L broth). The broth is clarified bycentrifugation and loaded onto an XAD-16 resin (Rohm and Haas) column (1kg XAD/1 g erythromycin analogs) at a flow rate of 2-4 mL/cm²-min. Theloaded resin is washed with 2 column volumes of 20% (v/v) ethanol inwater and the erythromycin analogs are eluted from the resin withacetone and collected in ½column volume fractions. The fractionscontaining erythromycin analogs are identified by thin-layerchromatography (ethyl acetate:hexanes 1:1) and HPLC/MS.

[0212] The acetone fractions containing erythromycin analogs are pooledand the volatiles are removed under reduced pressure. The resultingaqueous mixture is extracted with ethyl acetate. The ethyl acetateextract is washed with saturated NaH₂CO₃ and brine solutions, dried oversodium or magnesium sulfate, filtered, and concentrated to dryness underreduced pressure. Crude material is dissolved in dichloromethane andloaded onto a pad of silica gel and washed with dichloromethane:methanol(96:4 v/v) until the eluent is no longer yellow. The desired material iswith dichloromethane:methanol:triethylamine (94:4:2 v/v) and collectedin fractions. Fractions containing erythromycin are identified bythin-layer chromatography, collected and concentrated under reducedpressure. This material is recrystallized from dichloromethane/hexanes.

[0213] This general procedure is illustrated as follows, which isspecific for intermediate of compounds (1)-(3) or (1′)-(3′):

[0214] (i) 14-norerythromycins: 1 liter of ethanol was added to each of10 liters of fermentation broth. The broth was centrifuged and thesupernatant was passed through 0.6 liters of XAD (column dimensions 17cm×6.5 cm) at a flow rate of 100 mL/min. After loading, the column waswashed with 1.5 liters of 20% (v/v) ethanol in water. The desiredmaterial was then eluted with acetone. The fractions containing thismaterial were concentrated under reduced pressure until the volatileswere removed and the aqueous remainder was extracted with ethyl acetate.The ethyl acetate layers were washed with saturated sodium bicarbonatesolution, brine, dried with magnesium sulfate and concentrated underreduced pressure to give the crude extract.

[0215] Crude material (0.6 g) was dissolved in dichloromethane andgravity filtered through a 3 cm pad of silica gel in a 6 cm diameterfritted funnel. The material was eluted with 400 mL of dichloromethanefollowed by 400 mL dichloromethane:methanol:triethylamine (90:10:2 v/v)and collected in 40 mL fractions. Fractions containing erythromycin wereidentified by thin-layer chromatography (ether:methanol:NH₄OH 90:8:2v/v, Rf˜0.35 and dichloromethane:methanol 95:5 v/v, Rf˜0) andconcentrated under reduced pressure. This material was recrystallizedfrom dichloromethane/hexanes.

[0216] (ii) 15-methyl-erythromycins: 8 liters of ethanol was added toapproximately 80 liters of fermentation broth. The broth was centrifugedand the supernatant was passed through 2.5 liters of XAD at a flow rateof 230 mL/min. After loading the column was washed with 1 liter of waterand 5 liters of 20% (v/v) ethanol in water. The desired material wasthen eluted with acetone. The fractions containing this material wereconcentrated under reduced pressure until the volatiles were removed andthe aqueous remainder was extracted with ethyl acetate. The ethylacetate layers were washed with saturated sodium bicarbonate solution,brine, dried with magnesium sulfate and concentrated under reducedpressure to give the crude extract

[0217] Crude material (8.3 g) was dissolved in dichloromethane andgravity filtered through a 3 cm pad of silica gel in a 9 cm diameterfritted funnel. The material was eluted with 200 mL of dichloromethanefollowed by 600 mL of dichloromethane: methanol (96:4 v/v) followed by900 mL dichloromethane:methanol:triethylamine (89:9:2 v/v) and collectedin 40 mL fractions. Fractions containing erythromycin were identified bythin-layer chromatography (ether:methanol:NH₄OH 90:8:2 v/v, Rf˜0.4 anddichloromethane:methanol 95:5, Rf˜0.05) and concentrated under reducedpressure. This material was re-subjected to the above procedure beforeit was suitable for recrystallization.

[0218] (iii) 14-nor-6-deoxy-erythromycins: 1 liter of ethanol was addedto each of 2 10 liter fermenting. The broths were centrifuged and thesupernatants were combined for a total of approximately 22 liters. Thecombined broths were then passed through 1 liter of XAD (columndimensions 23.5 cm×6.5 cm (i.d.) at a flow rate of 170 mL/min. Afterloading the column was washed with 2 liters of 20% (v/v) ethanol inwater. The desired material was then eluted with acetone. The fractionscontaining this material were concentrated under reduced pressure untilthe volatiles were removed and the aqueous remainder was extracted withethyl acetate. The ethyl acetate layers were washed with saturatedsodium bicarbonate solution, brine, dried with magnesium sulfate andconcentrated under reduced pressure to give the crude extract.

[0219] (iv) 15-methyl-6-deoxy-erythromycins: 1 liter of ethanol wasadded to each of 3 fermentors containing 10 liters of broth. The brothswere centrifuged and the supernatant was passed over 1.25 liters of XAD(column dimensions 40 cm×6.5 cm) at a flow rate of 130 mL/min. Thecolumn was then washed with 3 liters of 20% (v/v) ethanol in water. Thedesired material was then eluted with acetone. The fractions containingthis material were concentrated under reduced pressure until thevolatiles were removed and the aqueous remainder was extracted withethyl acetate. The ethyl acetate layers were washed with saturatedsodium bicarbonate solution, brine, dried with magnesium sulfate andconcentrated under reduced pressure to give the crude extract.

[0220] Crude material (2.8 g) was dissolved in dichloromethane andgravity filtered through a 3 cm pad of silica gel in a 6 cm diameterfritted funnel. The material was eluted with 400 mL ofdichloromethane:methanol (96:4 v/v) followed by 400 mLdichloromethane:methanol:triethylamine (89:9:2 v/v) and collected in 40mL fractions. Fractions containing erythromycin were identified bythin-layer chromatography (ether:methanol:NH₄OH 90:8:2 v/v anddichloromethane:methanol 95:5) and concentrated under reduced pressure.This material required further purification by silica gelchromatography.

Example 4 Synthesis of 6-O-methyl-14-norerythromycin A. i.e., Formula(4) Where R_(a)=Me, G=R_(d)=Me, R_(c)=H, R_(e)=H

[0221] A. 14-Norerythromycin A 9-Oxime

[0222] A solution of 14-norerythromycin A (0.621 g, 80% pure),hydroxylamine (0.5 ml of 50% aqueous solution) and acetic acid (0.2 ml)in isopropanol (2 ml) was kept at 50° C. for 22 hours. It was extractedwith chloroform/ethanol (3/2), washed with sodium bicarbonate, brine,and dried over MgSO₄. Filtration and evaporation in vacuo yielded acrude product (0.65 g) as a white solid which was used directly for nexttransformation.

[0223] B. 14-Norerythromycin A-9-[O-(1-isopropoxycyclohexyl)]oxime

[0224] To a solution of above crude 14-noreythromycin A 9-oxime (0.65 g)and 1,1-diisopropoxy-cyclohexanone (0.95 ml) in methylene chloride (2ml) was added pyridinium p-toluenesulfonate (PPTS) (0.333 g) inmethylene chloride (2 ml). After stirring overnight, the mixture wasextracted (chloroform/ethanol 3:2), washed (NaHCO₃-H₂O, brine), anddried (MgSO₄). After filtration and evaporation in vacuo, the crudeproduct was repeatedly driven with toluene and isopropanol to yield 0.74g of product, which was used directly for next reaction.

[0225] C. 2′,4″-bis-O-trimethylsilyl-14-norerythiomycinA-9-[O-(1-isopropoxycyclohexyl)]oxime

[0226] To a solution of 14-norerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (0.74 g) in methylene chloride (6ml) was added a solution of trimethylsilyl imidazole (0.33 ml) andtrimethylsilyl chloride (0.18 ml) in methylene chloride (2 ml) at 0° C.After 5 minute stirring, ethyl acetate was added, washed (NaHCO₃-H₂O,brine), and dried (MgSO₄). Flash chromatography on silica gel (10:1hexanes:acetone, 1% triethylamine) afforded pure product as a whitesolid (0.50 g). Mass spectrometry reveals [M+H⁺]=1020.

[0227] D. 6-O-Methyl-2′,4″-bis-O-trimethylsilyl-14-norerythromycinA-9-[O-(1-isopropoxycyclohexyl)]oxime

[0228] A solution of 2′,4″-bis-O-trimethylsilyl-14-norerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (0.3 g, 0.29 mmol) in 1:1methylsulfoxide/tetrahydrofuran (DMSO/THF) (1.4 ml) was treated with 0.3ml of a 2 M solution of methyl bromide in ether and cooled to 10° C. Amixture of 1 M solution of potassium tert-butoxide in THF (0.6 ml) andDMSO (0.6 ml) was added over 6 hours using a syringe pump. The reactionwas then diluted with ethyl acetate, washed with saturated NaHCO₃,brine, and dried over MgSO₄. Filtration and evaporation in vacuo yieldeda crude product (0.29 g) as a white solid. Mass spectrometry reveals[M+H⁺]=1034.

[0229] E. 6-O-Methyl-14-norerythromycin A 9-oxime

[0230] A mixtureof6-O-methyl-2′,4″-bis-O-trimethylsilyl-14-norerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (0.29 g), acetic acid (3.6 ml),acetonitrile (6 ml) and water (3 ml) was stirred at ambient temperaturefor 4.5 hours. The mixture was driven to dryness using toluene to give acrude product as white solid (0.24 g), which was used directly for nextstep without further purification.

[0231] F. 6-O-Methyl-14-norerythromycin A

[0232] A mixture of6-O-methyl-14-norerythromycin A 9-oxime (0.24 g),sodium hydrosulfite (0.45 g, 85% pure), water (3 ml), ethanol (3 ml) andformic acid (0.07 ml) was kept at 85° C. for 8 hours. The reaction wasbrought to pH 8 with 1 N NaOH and extracted with ethyl acetate. Theorganic extract was washed with brine, dried over MgSO₄, filtered, andconcentrated to yield a crude product as a white solid (0.2 g). Massspectrometry reveals [M+H³⁰]=735.

Example 5 Synthesis of 6-O-methyl-14,15-dehydroerythromycin A, i.e.Formula (4) Where G=R_(d)=CH═CH₂, (R_(x)=R₃′=H), R₁=Me, R_(c)=H, R_(e)=H

[0233] A. 14,15-dehydroerythromycin A 9-oxime

[0234] A suspension of 14,15-dehydroerythromycin A (1.984 g, 47% purity,1.2 mmol) in 6 mL of 2-propanol was treated with 1.97 mL of 50% aqueoushydroxylamine and stirred until dissolved. Acetic acid (0.62 mL) wasadded and the mixture was stirred for 25 hours at 50° C. Upon cooling toambient temperature, saturated NaHCO₃ was added and the mixture wasconcentrated en vacuo to remove isopropanol. The resulting aqueousmixture was extracted three times with 250-mL portions of CHCl₃. Theorganic extracts were combined, washed with saturated NaHCO₃, water, andbrine, then dried over MgSO₄, filtered, and concentrated to yield 0.92 gof product.

[0235] B. 14,15-dehydroerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime

[0236] The oxime from (A) (0.92 g) was dissolved in 6.2 mL of CH₂Cl₂ andtreated with 1,1-diisopropoxycyclohexane (1.23 g) and pyridiniump-toluenesulfonate (0.464 gm) for 15 hours at ambient temperature. Themixture was diluted with 160 mL of CH₂Cl₂, then washed sequentially withsaturated NaHCO₃, water, and brine. The organic phase was dried withMgSO₄, filtered, and evaporated to yield a brown syrup. Chromatographyon silica gel (gradient from toluene to 1:1 toluene/acetone +1% Et₃N)yielded 0.998 g of product.

[0237] C. 2′,4″-bis(O-trimethylsilyl)-14,15-dehydroerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime

[0238] A solution of 14,15-dehydroerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (998 mg, 9.96) in 11.25 mL of CH₂Cl₂was cooled on ice under inert atmosphere and treated with a solution ofchlorotrimethylsilane (0.24 mL) and 1-trimethylsilylimidazole (0.44 mL).After 30 minutes, the reaction was diluted with 250 mL of ethyl acetateand washed sequentially with saturated NaHCO₃, water, and brine. Theorganic phase was dried with MgSO₄, filtered, and evaporated to yield1.002 g of product.

[0239] D2′,4″-bis(O-trimethylsilyl)-6-O-methyl-14,15-dehydroerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime

[0240] A solution of2′,4″-bis-O-trimethylsilyl-14,15-dehydroerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (1.00 g, 20.7 mmol) in 9.69 mL of1:1 tetrahydrofuran/methylsulfoxide was cooled to 10° C. and treatedwith 0.97 mL of 2.0 M methyl bromide in ether under inert atmosphere. Amixture of methylsulfoxide (1.94 mL) and 1.0 M potassium tert-butoxidein tetrahydrofuran (1.94 mL) was added slowly. The reaction wasmonitored by thin-layer chromatography (silica gel, 10:1toluene/acetone), and was judged complete after addition of 1.6 molarequivalents of base. The reaction was diluted with 200 mL of ethylacetate and 70 mL of saturated NaHCO₃. The mixture was transferred to aseparatory funnel, diluted with 850 mL of ethyl acetate and 280 mL ofsaturated NaHCO₃, then washed sequentially with water and brine. Theorganic phase was dried with MgSO₄, filtered through Celite, andevaporated to yield 21.2 g of crude6-O-methyl-2′,4″-bis-O-trimethylsilyl-14,15-dehydroerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime. This was carried on without furtherpurification.

[0241] E. 6-O-methyl-14,15-dehydroerythromycin A 9-oxime

[0242] A solution of6-O-methyl-2′,4″-bis-O-trimethylsilyl-14,15-dehydroerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (1.0 g) in 9.8 mL of 2:1acetonitrile/water was treated with 5.3 mL of acetic acid, and stirredfor 8 hours at ambient temperature. The mixture was concentrated envacuo, then repeatedly concentrated after addition of toluene to yield0.797 g of crude 6-O-methyl-14,15-dehydroerythromycin A 9-oxime.

[0243] F. 6-O-methyl-14,15-dehydroerythromycin A

[0244] A solution of 6-O-methyl-14,15-dehydroerythromycin A 9-oxime(0.797 g) and sodium hydrosulfite (85%, 1.02 g) in 7.5 mL of 1:1ethanol/water was placed under inert atmosphere. Formic acid (0.186 mL)was added dropwise, and the mixture was stirred at 80° C. for 3 hours.After cooling to ambient temperature, the reaction was adjusted to pH 10with 6 N NaOH and extracted three times with 150-mL portions of ethylacetate. The organic extracts were combined and washed sequentially withsaturated NaHCO₃, water, and brine. The organic phase was dried withMgSO₄, filtered, and evaporated to yield 0.68 g of6-O-methyl-14,15-dehydroerythromycin A suitable for further conversion.

Example 6 Synthesis of 6-O-methyl-15-methylerythromycin A, i.e., Formula(4) Where G=R_(d)=propyl, R_(a)=Me, R_(c)=H, R_(e)=H

[0245] A. 15-Methylerythromycin A 9-Oxime

[0246] A suspension of 15-methylerythromycin A (20.0 g, 85% purity, 22.6mmol) in 40 mL of 2-propanol was treated with 20.5 mL of 50% aqueoushydroxylamine and stirred until dissolved. Acetic acid (6.41 mL) wasadded and the mixture was stirred for 15 hours at 50° C. Upon cooling toambient temperature, saturated NaHCO₃ was added and the mixture wasconcentrated en vacuo to remove isopropanol. The resulting aqueousmixture was extracted three times with 250-mL portions of CHCl₃. Theorganic extracts were combined, washed with saturated NaHCO₃, water, andbrine, then dried over MgSO₄, filtered, and concentrated to yield 20.5 gof crude product. Analysis by LC/MS revealed a 94:6 mixture of E and Zoximes, [M+H]⁺=764.

[0247] B. 15-Methylerythromycin A 9-[O-(1-isopropoxycyclohexyl)]oxime

[0248] The crude oxime from above (20.5 g) was dissolved in 55 mL ofCH₂Cl₂ and treated with 1,1-diisopropoxycyclohexane (27.3 mL) andpyridinium p-toluenesulfonate (9.8 gm) for 15 hours at ambienttemperature. The mixture was diluted with 160 mL of CH₂Cl₂, then washedsequentially with saturated NaHCO₃, water, and brine. The organic phasewas dried with MgSO₄, filtered, and evaporated to yield a brown syrup.Chromatography on silica gel (gradient from 2:1 to 3:2hexanes/acetone+1% Et₃N) yielded 18.0 g of product.

[0249] C. 2′, 4″-bis-O-trimethylsilyl-15-methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime

[0250] A solution of 15-Methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (9.00 g, 9.96 mmol) in 25 mL ofCH₂Cl₂ was cooled on ice under inert atmosphere and treated with asolution of chlorotrimethylsilane (1.89 mL) and1-trimethylsilylimidazole (3.65 mL) in 8 mL of CH₂Cl₂. After 30 minutes,the reaction was diluted with 250 mL of ethyl acetate and washedsequentially with saturated NaHCO₃, water, and brine. The organic phasewas dried with MgSO₄, filtered, and evaporated. The crude product waspurified by silica gel chromatography (gradient from hexanes to 10:1hexanes/acetone+1% Et₃N), yielding 7.8 g of product.

[0251] D. 6-O-Methyl-2′, 4″-bis-O-trimethylsilyl-15-methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime

[0252] A solution of 2′,4″-bis-O-trimethylsilyl-15-methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (21.7 g, 20.7 mmol) in 41.4 mL oftetrahydrofuran was cooled to 10° C. and treated with 41.4 mL ofmethylsulfoxide and 20.7 mL of 2.0 M methyl bromide in ether under inertatmosphere. A mixture of methylsulfoxide (41.4 mL) and 1.0 M potassiumtert-butoxide in tetrahydrofuran (41.4 mL) was added at a rate of ca. 20mL per hour. The reaction was monitored by thin-layer chromatography(silica gel, 10:1 toluene/acetone), and was judged complete afteraddition of 1.6 molar equivalents of base. The reaction was diluted with200 mL of ethyl acetate and 70 mL of saturated NaHCO₃. The mixture wastransferred to a separatory funnel, diluted with 850 mL of ethyl acetateand 280 mL of saturated NaHCO₃, then washed sequentially with water andbrine. The organic phase was dried with MgSO₄, filtered through Celite,and evaporated to yield 21.2 g of crude6-O-methyl-2′,4″-bis-O-trimethylsilyl-15-methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime. This was carried on without furtherpurification.

[0253] E. 6-O-Methyl-15-methylerythromycin A 9-oxime

[0254] A solutionof6-O-methyl-2′,4″-bis-O-trimethylsilyl-15-methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (21.2 g) in 110 mL of acetonitrilewas treated with 55 mL of water and 67 mL of acetic acid, and stirredfor 8 hours at ambient temperature. The mixture was concentrated envacuo, then repeatedly concentrated after addition of toluene to yield19.7 g of 6-O-methyl-15-methylerythromycin A 9-oxime.

[0255] F. 6-O-Methyl-15-methylerythromycin A

[0256] A solution of 6-O-methyl-15-methylerythromycin A 9-oxime (19.7 g)and sodium hydrosulfite (85%, 23.1 g) in 280 mL of 1:1 ethanol/water wasplaced under inert atmosphere. Formic acid (3.75 mL) was added dropwise,and the mixture was stirred at 80° C. for 4.5 hours. After cooling toambient temperature, the reaction was treated with saturated NaHCO₃ andextracted three times with 400-mL portions of ethyl acetate. The organicextracts were combined and washed sequentially with saturated NaHCO₃,water, and brine. The organic phase was dried with MgSO₄, filtered, andevaporated to yield 15.1 g of 6-O-methyl-15-methylerythromycin Asuitable for further conversion.

Example 7 Synthesis of5-O-(2′-acetyldesosaminyl)-10,11-anhydro-3-deoxy-3-oxo-6-O-methyl-14-norerythronolideA (Anhydro form of Formula (6), R_(a)=Me, G=R_(d)Me, R_(c)=Ac, R_(b)=H)

[0257] A. 5-O-Desosaminyl-6-O-methyl-14-norerythronolide A

[0258] A mixture of 6-O-methyl-14-norerythromycin A (77 mg, crude),0.073 ml of 12 N HCl and water (2 ml) was stirred at ambient temperaturefor 3 hours. The mixture was brought to pH 8 with 8 N KOH, and extractedwith ethyl acetate. The organic extract was washed with brine, driedwith MgSO₄, filtered, and evaporated. The residue was chromatographed onsilica gel (3:1/hexanes: acetone, 1% triethylamine) to give pure productas a white solid (42 mg). Mass spectrometry reveals [M+H³⁰]=576.

[0259] B. 5-O-(2′-Acetyldesosaminyl)-6-O-methyl-14-norerythronolide A: Amixture of 5-O-desosaminyl-6-O-methyl-14-norerythronolide A (73 mg),potassium carbonate (20 mg), acetic anhydride (14w1) and acetone (1 ml)was stirred at ambient temperature for 18 hours. Ethyl acetate wasadded, washed with water and brine, dried over MgSO₄, filtered, andevaporated. The residue was chromatographed on silica gel(3:1/hexanes:acetone, 1% triethylamine) to yield the pure product (71mg) as a white solid. Mass spectrometry reveals [M+H³⁰]=618.

[0260] C. 5-O-(2′-Acetyldesosaminyl-3-deoxy-3-oxo-6-O-methyl-14-norerythronolide A (Formula (6), R_(a)=Me,G=R_(d)Me, R_(b)=H, R_(c)=Ac)

[0261] A solution of5-O-(2′-acetyldesosaminyl)-6-O-methyl-14-norerythronolide A (99 mg) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(EDC) hydrochloride (206mg) in dichloromethane (2 ml) was treated with DMSO (0.21 ml) and cooledto 5° C. A solution of pyridinium trifluoroacetate (208 mg) indichloromethane (2 ml) was added via a syringe pump in 4 hours. Ethylacetate was then added, washed with saturated NaHCO₃, water, brine, anddried over MgSO₄, filtered, and evaporated. The residue waschromatographed on silica gel (3:1/hexanes:acetone, 1% triethylamine) toyield the pure product (94 mg) as a white solid. Mass spectrometryreveals [M+H³⁰]=616.

[0262] D.5-O-(2′-Acetyldesosaminyl)-3-deoxy-3-oxo-11-O-methanesulfonyl-6-O-methyl-14-norerythronolideA

[0263] To a solution of5-O-(2′-acetyldesosaminyl)-3-deoxy-3-oxo-6-O-methyl-14-norerythronolideA (93 mg) in dry pyridine (1 ml) was added methanesulfonyl chloride(0.057 ml) at 5° C. After 3 hours at 5° C., the reaction was warmed toambient temperature and kept for an additional 15 hours. The mixture wasdiluted with ethyl acetate, washed with saturated NaHCO₃(2×), water(3×), brine, and dried over MgSO₄, filtered, and evaporated. The residuewas chromatographed on silica gel (2:1/hexanes:acetone, 1%triethylamine) to yield the pure product (72 mg) as a white solid. Massspectrometry reveals [M+H³⁰]=695.

[0264] E.5-O-(2′-Acetyldesosaminyl)-10,11-anhydro-3-deoxy-3-oxo-6-O-methyl-14-norerythronolideA

[0265] A solution of5-O-(2′-acetyldesosaminyl)-3-deoxy-3-oxo-11-O-methanesulfonyl-6-O-methyl-14-norerythronolideA (73 mg) in acetone 1ml) was treated with diazabicycloundecene (32 μl)at ambient temperature for 18 hours. The mixture was diluted with ethylacetate, washed with saturated NaHCO₃, water, brine, and dried overMgSO₄, filtered, and evaporated. The residue was chromatographed onsilica gel (2:1/hexanes:acetone, 1% triethylamine) to yield the pureproduct (50 mg) as a white solid. Mass spectrometry reveals [M+H³⁰]=598.

Example 8 Synthesis of2′-O-Benzoyl-6-O-methyl-3-descladinosyl-3-oxo-10,11-anhydro-14,15-dehydroerythromycinA (Anhydro form of Formula (6) G=R_(d)=allyl, R_(a)=Me, R_(b)=H,R_(c)=Benzoyl)

[0266] A. 2′-O-Benzoyl-6-O-methyl-14,15-dehydroerythromycin A

[0267] A solution of 6-O-methyl-14,15-dehydroerythromycin A (668 mg),benzoic anhydride (385 mg), and triethylamine (0.25 mL) in 3.6 mL ofCH₂Cl₂ was stirred for 2 days. After addition of saturated NaHCO₃, themixture was extracted three times with CH₂Cl₂. The organic extracts werecombined and evaporated to dryness, and the product was purified bysilica chromatography (90:9:1 toluene/acetone/Et₃N) to give 477 mg ofproduct; LC-MS shows [M+H]⁺=850.6.

[0268] B.2′-O-Benzoyl-6-O-methyl-4′,11-bis(O-methanesulfonyl)-14,15-dehydroerythromycinA

[0269] A solution of 2¹-O-benzoyl-6-O-methyl-14,15-dehydroerythromycin A(549 mg) and methanesulfonyl chloride (0.50 mL) in 2.39 mL of pyridinewas stirred for 24 hours, then diluted with CH₂Cl₂ and saturated NaHCO₃.The mixture was extracted three times with CH₂Cl₂. The organic extractswere combined and evaporated to dryness, and the product was purified bysilica chromatography (90:9:1 toluene/acetone/Et₃N) to give 530 mg ofproduct; LC-MS shows [M+H]⁺=1006.5.

[0270] C.2′-O-Benzoyl-6-O-methyl-4″-O-methanesulfonyl-10,11-anhydro-14,15-dehydroerythromycinA

[0271] A mixture of 2′-O-benzoyl-6-O-methyl-4′,11-bis(O-methanesulfonyl)14,15-dehydroerythromycin A (59 mg) and diazabicycloundecene (0.018 mL)in 0.195 mL of acetone was stirred for 24 hours, then dried in vacuo.The product was purified by silica chromatography (90:9:1toluene/acetone/Et₃N) to give 50 mg of product; LC-MS shows[M+H]⁺=910.5.

[0272] D.2′-O-Benzoyl-6-O-methyl-3-descladinosyl-10,11-anhydro-14,15-dehydroerythromycinA

[0273] A mixture of2′-O-benzoyl-6-O-methyl-4″-O-methanesulfonyl-10,11-anhydro-14,15-dehydroerythromycinA (337 mg), 1.5 mL of acetonitrile, and 6.9 mL of 3 N HCl was stirredfor 22 hours. The acetonitrile was removed in vacuo, the pH of theaqueous residue was adjusted to 12 by addition of NaOH, and the productwas extracted using 4 portions of CH₂Cl₂. The combined extracts weredried and evaporated. The product was purified by silica chromatography(gradient from 96:4 CH₂Cl₂/MeOH to 95:4:1 CH₂Cl₂/MeOH/Et₃N) to give 197mg, [M+H]⁺=674.4.

[0274] E.2′-O-Benzoyl-6-O-methyl-3-descladinosyl-3-oxo-10,11-anhydro-14,15-15dehydroerythromycin A

[0275] A suspension of2′-O-benzoyl-6-O-methyl-3-descladinosyl-10,11-anhydro-14,15-dehydroerythromycinA (226 mg) and the Dess-Martin periodinane (427 mg) in 14.6 mL of CH₂Cl₂(14.6 mL) was stirred for 1 hour. The mixture was diluted with CH₂Cl₂and saturated NaHCO₃. The product was extracted using 3 portions ofCH₂Cl₂, and the extracts were combined, dried, and evaporated. Silicagel chromatography (90:9:1 toluene/acetone/Et₃N) yielded the product,168 mg. [M+H]⁺=672.4. ¹³C-NMR (CDCl₃, 100 MHz): δ206.78, 203 (br),168.19, 165.08, 141.36, 139.58, 132.74, 131.51, 130.46, 129.79, 128.25,120.18, 102.09, 80.79, 80.40, 78.70, 72.52, 71.91, 69.19, 63.76, 51.10,50.54, 47.08, 40.73, 39.87, 37.77, 31.23, 22.13, 20.98, 18.52, 14.28,14,15, 13.55.

Example 9 Synthesis of5-O-(2′-acetyldesosaminyl)-10,11-anhydro-3-deoxy-3-oxo-6-O-methyl-15-methylerythronolideA (Anhydro form of Formula (6); R_(a)=Me, G=R_(d)=propyl, R_(b)=H,R_(c)=Ac)

[0276] A. 6-O-methyl-3-descladinosyl-15-methylerythromycin A

[0277] A mixture of 6-O-methyl-15-methylerythromycin A (15.1 g) and 280mL of 0.5 N HCl was stirred at ambient temperature for 3 hours. The pHwas adjusted to 9 by addition of 6 N NaOH, and the resulting precipitatewas collected by vacuum filtration, washed with water, and dried. Thefiltrate was extracted three times with 400-mL portions of ethylacetate. The organic extracts were combined, washed sequentially withsaturated NaHCO₃, water, and brine, then dried over MgSO₄, filtered, andevaporated to provide further product. The combined crude products werechromatographed on silica gel to yield 9.35 g of pure6-O-methyl-3-descladinosyl-15-methylerythromycin A. ES-LC/MS shows[M+H]⁺=605.

[0278] B. 2′-O-Acetyl-6-O-methyl-3-descladinosyl-15-methylerythromycin A

[0279] A solution of acetic anhydride (2.92 mL) in 35 mL of ethylacetate was added dropwise to a solution of6-O-methyl-3-descladinosyl-15-methylerythromycin A (9.35 g) in 40 mL ofethyl acetate. The mixture was stirred for 30 minutes after completionof addition, then concentrated. Chromatography on silica gel (2:1hexanes/acetone) gave 8.35 g of2′-O-acetyl-6-O-methyl-3-descladinosyl-15-methylerythromycin A. ES-LC/MSshows [M+H]⁼=647.

[0280] C.2′-O-Acetyl-6-O-methyl-3-descladinosyl-3-oxo-15-methylerythromycin A

[0281] A solution of2′-O-acetyl-6-O-methyl-3-descladinosyl-15-methylerythromycin A (8.3 g)and 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (16.51 g)in 64 mL of dichloromethane and 15.47 mL of methylsulfoxide was placedunder inert atmosphere and cooled on ice. A solution of pyridiniumtrifluoroacetate (16.63 g) in 64 mL of dichloromethane was added at arate such that addition would be complete in 4 hours, and the reactionwas monitored by thin-layer chromatography. Complete reaction wasobserved after addition of 73% of the solution, and so the reaction wasthen quenched by addition of 600 mL of ethyl acetate and 200 mL ofsaturated NaHCO₃. The organic layer was collected and washedsequentially with saturated NaHCO₃, water, and brine, then dried overMgSO₄, filtered, and evaporated to yield 8.4 g of crude product.Chromatography on silica gel (3:1 hexanes/acetone) gave 6.75 g of2′-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-15-methylerythromycin A.ES-LC/MS shows [M+H]⁼=645.

[0282] D.2′-O-Acetyl-6-O-methyl-3-descladinosyl-3-oxo-11-O-methanesulfonyl-15-methylerythromycinA

[0283] Methanesulfonylchloride (5.68 mL) was added dropwise to asolution of2′-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-15-methylerythromycin A(6.73 g) in 35 mL of pyridine at 0° C. The mixture was brought toambient temperature and quenched by addition of 700 mL of ethyl acetateand 200 mL of saturated NaHCO₃. The organic layer was collected andwashed sequentially with saturated NaHCO₃, water, and brine, then driedover MgSO₄, filtered, and evaporated to yield 8.2 g of crude product.Chromatography on silica gel (5:2 hexanes/acetone) gave 5.04 g of2′-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-11-O-methyl-anesulfonyl-15-methylerythromycinA. ES-15 LC/MS shows [M+H]⁼=723.

[0284] E.2′-O-Acetyl-6-O-methyl-3-descladinosyl-3-oxo-10,11-anhydro-15-methylerythromycinA

[0285] 1,8-Diazabicyclo[5.4.0]undec-7-ene (5.22 mL) was added dropwiseto a solution of2′-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-11-O-methyl-anesulfonyl-15-methylerythromycinA (5.03 g) in 23 mL of acetone. The solution was concentrated after 4.5hours, and the residue was chromatographed on silica gel (5:2hexanes/acetone) to give 3.72 g of2′-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-10,11-anhydro-15-methylerythromycinA. ES-LC/MS shows [M+H]⁻=627.

Example 10 Synthesis of5-O-(2′-acetyldesosaminyl)-10,11-anhydro-3,6-dideoxy-3-oxo-15-methylerythronolideA (Formula (6), anhydro form, G=R_(d)=propyl, OR_(a) replaced by H,R_(b)=H, R_(c)=Ac)

[0286] To a solution of 6-deoxy-15-methyl erythromycin C (220 mg, 0.307mmol) in dichloromethane (5 mL) were given potassium carbonate (50 mg)and acetic anhydride (100 L, 0.9 mmol), and the reaction was stirred atroom temperature for 16 hours. The solution was filtered, sodiumhydroxide (1N, 25 mL) and brine (25 mL) added and the aqueous layer wasextracted with ethyl acetate 6 times. The combined organic layers weredried with sodium sulfate, filtered, and the solvent removed in vacuo.The crude product the 2′ acetylated form of the starting material wascarried on to the next step.

[0287] The crude product was dissolved in pyridine (5 mL) and mesylchloride (70 L, 0.9 mmol) was added. The reaction was stirred at −20° C.for 2 days, poured on sodium hydroxide (1 N, 25 mL) and brine (25 mL)and the aqueous layer was extracted with ethyl acetate 6 times. Thecombined organic layers were dried with sodium sulfate, filtered, andthe solvent removed in vacuo. The residue was purified by chromatographyon silica gel (toluene/acetone=3:1, 1% ammonium hydroxide) to yield11,4″-dimesylated form (190 mg, 68% over two steps).

[0288] The 11, 4″-dimesylated form (190 mg, 0.21 mmol) was dissolved inacetone (7 mL) and DBU (63 L, 0.42 mmol) was added, and the reaction wasstirred at room temperature over night. The mixture was poured on sodiumhydroxide (1 N, 25 mL) and brine (25 mL) and the aqueous layer wasextracted with ethyl acetate 6 times. The combined organic layers weredried with sodium sulfate, filtered, and the solvent removed in vacuo.The crude product, the 10,11-dehydro form of6-deoxy-15-methylerythromycin was carried on to the next step.

[0289] To the crude product from the above step was added hydrochloricacid (30 mL, 3 N) and ethanol (2 mL) and the mixture was stirredvigorously for 6 hours. Sodium hydroxide (5 mL, 10 N) was added and theaqueous layer was extracted with ethyl acetate 6 times. The combinedorganic layers were dried with sodium sulfate, filtered, and the solventremoved in vacuo. The crude product, the anhydro form of formula (1)(but with OH at position 3) where G=R_(d)=propyl, OR_(a) is replaced byH, R_(b)=R_(c)=H, was carried on to the next step.

[0290] To the crude product from the above step in dichloromethane (5mL) was added acetic anhydride (50 L, 0.45 mmol) and potassium carbonate(100 mg) and the mixture was stirred vigorously for 9 hours. Thereaction was filtered, sodium hydroxide (20 mL, 1 N) and brine (25 mL)were added and the aqueous layer was extracted with ethyl acetate 6times. The combined organic layers were dried with sodium sulfate,filtered, and the solvent removed in vacuo. The residue was purified bychromatography on silica gel (toluene/acetone=3:1, 1% ammoniumhydroxide) to yield the 2′ acetylated form of the starting material (110mg, 89% over three steps).

[0291] The product of the above step (110 mg, 0.184 mmol) was dissolvedin dichloromethane (10 mL) and Dess-Martin reagent (220 mg, 0.53 mmol)was added. The reaction was stirred at room temperature for 45 min. Thereaction was quenched with Sodium hydroxide (20 mL, 1 N) and brine (25mL) and the aqueous layer was extracted with ethyl acetate 6 times. Thecombined organic layers were dried with sodium sulfate, filtered, andthe solvent removed in vacuo. The residue was purified by chromatographyon silica gel (toluene/acetone, gradient =6:1-3:1, 1% ammoniumhydroxide) to yield the compound of formula (6), anhydro form, whereG=R_(d)=propyl, OR_(a) is replaced by H, R_(b)=H, R_(c)=Ac (94 mg, 86%).

Example 11 Compound of Formula (4): R_(a)=allyl, G=R_(d)=Me

[0292] Step 1. Allylation of Intermediate Antibiotic at 6-OH: A solutionof 2′,4″-bis-O-trimethylsilyl-14-norerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime, Formula (I), (R_(a) is OH, G=R_(d)is methyl, protected at 2′ and 4″ with trimethylsilyl and at C9=O by theisoproxycyclohexyl oxime)) (7.8 g, 7.44 mmol) in 30 mL oftetrahydrofuran was cooled on ice and treated with 30 mL ofmethylsulfoxide and 2.58 mL of freshly distilled allyl bromide underinert atmosphere. A mixture of methylsulfoxide (29.8 mL) and 1.0 Mpotassium tert-butoxide in tetrahydrofuran (29.8 mL) was added at a rateof 1.33 molar equivalents of base per hour. The reaction was monitoredby thin-layer chromatography (silica gel, 10:1 toluene/acetone), and wasjudged complete after addition of 3.6 molar equivalents of base. Thereaction was diluted with 700 mL of ethyl acetate and washedsequentially with saturated NaHCO₃, water, and brine. The organic phasewas dried with MgSO₄, filtered, and evaporated to yield 8.08 g of crude6-O-allyl-2′,4″-bis-O-trimethylsilyl-15-methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime. This was carried on without furtherpurification.

[0293] Step 2: A solution of6-O-allyl-2′,4″-bis-O-trimethylsilyl-15-methylerythromycin A9-[O-(1-isopropoxycyclohexyl)]oxime (8.08 g) in 42 mL of acetonitrilewas treated with 21 mL of water and 24 mL of acetic acid, and stirredfor 18 hours at ambient temperature. The mixture was concentrated afteraddition of 2-propanol, then repeatedly after addition of toluene toyield 7.7 g of crude product. Chromatography on silica gel (gradientfrom 2:1 to 1:1 hexanes/acetone+1% Et₃N) gave 3.75 g of6-O-allyl-15-methylerythromycin A 9-oxime.

[0294] Step 3: A solution of 6-O-allyl-15-methylerythromycin A 9-oxime(3.75 g) and sodium hydrosulfite (85%, 5.37 g) in 66 mL of 1:1ethanol/water was placed under inert atmosphere. Formic acid (0.845 mL)was added dropwise, and the mixture was stirred at 80° C. for 3.5 hours.After cooling to ambient temperature, the reaction was adjusted to pH 10with 6 N NaOH and extracted three times with 150-mL portions of ethylacetate. The organic extracts were combined and washed sequentially withsaturated NaHCO₃, water, and brine. The organic phase was dried withMgSO₄, filtered, and evaporated to yield 3.42 g of6-O-allyl-15-methylerythromycin A suitable for further conversion.

[0295] Other embodiments: In a similar manner, compounds of formula (4)wherein Y and Z are, together, ═O, R_(a) is allyl, is prepared from anintermediate where G is butyl, benzyl, vinyl, or 3-hydroxybutyl.

Example 12 Conversion to Formula (4) to Formula (6)

[0296] Step 1. A mixture of the compound prepared in Example 11 (77 mg,crude), 0.073 ml of 12 N HCl and water (2 ml) was stirred at ambienttemperature for 3 hours. The mixture was brought to pH 8 with 8 N KOH,and extracted with ethyl acetate. The organic extract was washed withbrine, dried with MgSO₄, filtered, and evaporated. The residue waschromatographed on silica gel (3:1/hexanes:acetone, 1% triethylamine) togive pure product as a white solid (42 mg).

[0297] Step 2. To protect the 2′ OH, a mixture the above compound (73mg), potassium carbonate (20 mg), acetic anhydride (14 μl) and acetone(1 ml) was stirred at ambient temperature for 18 hours. Ethyl acetatewas added, washed with water and brine, dried over MgSO₄, filtered, andevaporated. The residue was chromatographed on silica gel(3:1/hexanes:acetone, 1% triethylamine) to yield the pure product (71mg) as a white solid.

[0298] Step 3. A solution of the compound resulting from step 2 (99 mg)and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) hydrochloride(206 mg) in dichloromethane (2 ml) was treated with DMSO (0.21 ml) andcooled to 5° C. A solution of pyridinium trifluoroacetate (208 mg) indichloromethane (2 ml) was added via a syringe pump in 4 hours. Ethylacetate was then added, washed with saturated NaHCO₃, water, brine, anddried over MgSO₄, filtered, and evaporated. The residue waschromatographed on silica gel (3:1/hexanes:acetone, 1% triethylamine) toyield the pure compound of formula (6) (94 mg, R_(a) is allyl, R_(a) isacetate and G=R_(d) is CH₃).

[0299] Step 4. To deprotect 2′ OH, a solution of the compound resultingfrom step 3 (94 mg) in 5 mL methanol was stirred at room temperature for24 hours. The solvent was removed in vacuo to give the desired compoundof formula (6) (R_(a) is allyl, R_(a) is H, and G=R_(d) is CH₃).

[0300] Other embodiments: In a similar manner, compounds of formula (4)wherein R_(a) is allyl, R_(a) is H, and G is propyl, butyl, benzyl,vinyl, or 3-hydroxybutyl is prepared.

Example 13 Preparation of Compounds of Formula (5)

[0301] Step 1. Protection of 2′-OH

[0302] A solution of the compound from Example 12, Step 1 (1 mmol), isdissolved in dichloromethane (5 mL) and treated with benzoic anhydride(1.6 mmol) and triethylamine (1.6 mmol) for 26 hours at ambienttemperature. Aqueous 5% sodium carbonate is added and the mixture isstirred for 20 minutes, then extracted with dichloromethane. The extractis washed sequentially with sat. aq. NaHCO3 and brine, dried over MgSO4,filtered, and evaporated. The product is isolated by silica gelchromatography.

[0303] Step 2. Formation of 2,3-alkene and 11,12-carbamate

[0304] A solution of the compound from Step 1 (1 mmol) and1,1-carbonyldiimidazole (5 mmol) in 20 mL of 2:1tetrahydrofuran/dimethylformamide is cooled to −40°C., and a 1 Msolution of sodium bis(trimethylsilyl)amide in tetrahydrofaran (4.5 mL)is added slowly over 30 minutes. Stirring is continued for an additional2.5 hours at −40° C., then the mixture is warmed to ambient temperatureand kept overnight. A 0.5 M solution of NaH2PO4 (15 mL) is added, andthe mixture is extracted with ethyl acetate. The extract is dried overMgSO4, filtered, and evaporated. The crude material is dissolved in 15mL of 10:1 acetonitrile/tetrahydrofuran and treated with 2.5 mL ofconcentrated aqueous NH₃. After stirring overnight, the mixture isconcentrated to dryness and the residue is dissolved in ethyl acetateand washed with brine, dried over MgSO4, filtered, and evaporated.Silica gel chromatography yields the compound of Formula (5) whereR_(c)=PhCO.

[0305] Step 3. Removal of 2′-protection

[0306] The compound of Formula (5), R_(c) =PhCO, is dissolved inmethanol and heated at reflux for 5 hours to give the compound ofFormula (5), where R_(c)=H.

Example 14 Preparation of Compounds of Formula (5) for Making Compounds(101)-(103) of the Invention

[0307] Any of the compounds prepared above can be protected at the 2′position, treated with acid and dehydrated, then deprotected to obtainthe compound of formula (5), as shown in FIG. 2, wherein R_(c) is H,R_(a) is allyl, and G is R_(x)—CH═C(R_(d)′)—. Similarly, compounds offormula (6) wherein G is R_(x)—CH═C(R_(d)′)— and R_(d)′ is H, methyl,ethyl, propyl, butyl, benzyl, or 3-hydroxybutyl, are prepared asdescribed above using as starting material the compounds of formula (I)wherein R_(d)′ is as set forth above to make intermediates of compounds(101)-(103) of the invention.

Example 15 Conversion of ═O at Position 9 to ═NOH

[0308] According to the procedure of Example 6A, the carbonyl atposition 9 of erythromycins are converted to the corresponding oximes.

[0309] To a solution of any of the compounds prepared above (0.2 mmol)in ethanol is added hydroxylamine hydrochloride (76 mg, 1.1 mmol) andtriethylamine (56 μL, 0.4 mmol). The reaction mixture is stirredovernight at 80° C. and concentrated, and the residue taken up in ethylacetate. The organic phase is washed with aqueous 5% sodium bicarbonateand brine, dried over sodium sulfate, and concentrated in vacuo.Chromatography on silica gel (95:5:0.5 dichloromethane-methanol-ammonia)gives the corresponding E and Z oximes.

Example 16 Preparation of Compound of Formula (1): R_(a)═—CH₂CH═CH₂,R_(c) is H

[0310] A. Step 1. Protection at 2′-OH to form intermediate compound ofcompound (6) having hydroxyl group at C-3, R_(a) is allyl and R_(c) isbenzoyl

[0311] To a solution of the product of Example 12 or other embodimentthereof wherein G is propyl, butyl, benzyl, vinyl or 3-hydroxybutyl(2.49 g, 4.05 mmol) in dichloromethane (20 mL) is added benzoicanhydride (98%, 1.46 g, 6.48 mmol) and triethylamine (0.90 mL, 6.48mmol) and the white suspension is stirred for 26 hours at ambienttemperature. Aqueous 5% sodium carbonate is added and the mixture isstirred for 20 minutes. The mixture is extracted with dichloromethane.The organic phase is washed with aqueous 5% sodium bicarbonate andbrine, dried over sodium sulfate and concentrated in vacuo to give awhite foam. Chromatography on silica gel (30% acetone-hexanes) gives theprotected compound.

[0312] Step 2. Oxidation to form compound (6), R_(a) is allyl, R_(c) isbenzoyl

[0313] To a −10° C. solution under N₂ of N-chlorosuccinimide (0.68 g,5.07 mmol) in dichloromethane (20 mL) is added dimethylsulfide (0.43 mL,5.92 mmol) over 5 minutes. The resulting white slurry is stirred for 20minutes at −10° C. and then a solution of the compound resulting fromstep 1 (2.43 g, 3.38 mmol) in dichloromethane (20 mL) is added and thereaction mixture is stirred for 30 minutes at −10 to −5° C.Triethylamine (0.47 mL, 3.38 mmol) is added dropwise over 5 minutes andthe reaction mixture is stirred for 30 minutes at 0° C. The reactionmixture is extracted with dichloromethane. The organic phase is washedtwice with aqueous 5% sodium bicarbonate and once with brine, dried oversodium sulfate, and concentrated in vacuo to give a white foam.Chromatography on silica gel (30% acetone-hexanes) gives the oxidizedcompound.

[0314] Step 3: Form cyclic carbonate compound of formula (17) from FIG.6: R_(a) is —CH₂CH═CH₂, R_(c) is benzoyl.

[0315] To a −35° C. solution under nitrogen in THF (60 mL) of thecompound prepared in step 2 (3.58 g, 5.00 mmol) is added sodiumhexamethyldisilazide (1.0 M in THF, 5.5 mL, 5.5 mmol) and the resultingwhite suspension is stirred for 30 minutes. A solution ofcarbonyldiimidazole (4.05 g, 25 mmol) in THF (40 mL) is added dropwiseover 20 minutes at −35° C. and then the cold bath is removed and thereaction mixture is stirred for 30 minutes. The reaction mixture istaken up in ethyl acetate and washed with aqueous 5% sodium bicarbonateand brine, dried over sodium sulfate, filtered, and concentrated invacuo. Chromatography on silica gel (30% acetone-hexane) gives thedehydrated compound (2.6 g) as a white foam.

[0316] Step 4: Preparation to form 10,11 anhydro form of intermediatecompound (6): R_(a) is —CH₂CH═CH₂, R_(c) is benzoyl

[0317] To a solution of the compound in step 3, (2.59 g, 3.48 mmol) inbenzene (100 mL) is added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 5.0mL, 34 mmol). The reaction mixture is flushed with nitrogen, warmed to80° C., and stirred for 3.5 hours. The reaction mixture is cooled to 0°C. and aqueous 0.5 M NaH₂PO₄ (100 mL) is added. The mixture is extractedtwice with ethyl acetate and the combined organic layers are washed withbrine, dried over sodium sulfate and concentrated in vacuo to give awhite foam. Chromatography on silica gel (30% acetone-hexanes) gives thecompound.

[0318] Step 5: Derivation of Position 12 hydroxyl compound (7) from FIG.5: R_(a) is —CH₂CH═CH₂, R_(c) is benzoyl

[0319] A solution in THF (30 mL) of the compound prepared in step 4(1.74 g, 2.49 mmol) is cooled to −10° C. and flushed with nitrogen.Sodium hydride (80% in mineral oil, 150 mg, 5.00 mmol) is added and thereaction mixture is stirred for 10 minutes. A solution ofcarbonyldiimidazole (1.22 g, 7.50 mmol) in THF (20 mL) is added over 10minutes at −10° C. The cold bath is removed and the reaction mixture isstirred for 1 hour. The reaction mixture was extracted with ethylacetate and the organic phase is washed with aqueous 5% sodiumbicarbonate and brine, dried over sodium sulfate, and concentrated invacuo to give a white foam. Chromatography on silica gel (30%acetone-hexanes) gives the compound.

[0320] Step 6: Form cyclic carbamate compound (10) from FIG. 5: A, B, Dand E are H, R_(a) is —CH₂CH═CH₂, R_(c) is benzoyl

[0321] To a solution under nitrogen of a compound of formula (7) (R_(a)is allyl, R_(c) is benzoyl, 385 mg, 0.485 mmol), prepared as in step 5,in acetonitrile is added ethylenediamine (291 mg. 4.85 mmol) and thereaction mixture is stirred for 67 hours. The reaction mixture isextracted with ethyl acetate and the organic phase is washed withaqueous 5% sodium bicarbonate and brine, dried over sodium sulfate, andconcentrated in vacuo to give the title compound as colorless oil whichis used without further purification.

[0322] Step 7: Cyclization and Deprotection to Form Compound of Formula(1): R_(a) is —CH₂CH═CH₂, R_(c) is H

[0323] The crude oil prepared in step 6 is dissolved in methanol (5 mL),acetic acid (60 μL) is added, and the reaction mixture is stirred for 15hours at ambient temperature. The reaction mixture was extracted withethyl acetate and the organic phase is washed with aqueous 5% sodiumbicarbonate and brine, dried over sodium sulfate, and concentrated invacuo to give a slightly yellow glass. Chromatography on silica gel(95:5:0.5 dichloromethane-methanol-ammonia) gives the title compound asa white foam.

[0324] B. Alternate Steps 1′-3′ for Steps 1-5 in Example 16A to FormCompounds with Two Cladinose Moieties

[0325] Step 1′: Preparation of Intermediate Compound (4) from FIG. 4:R_(a) is —CH₂CH═CH₂, R_(c) and R_(e) are acetyl

[0326] To a sample of the compound from Example 11, step 3 or anembodiment thereof wherein G is propyl, butyl, benzyl, vinyl or3-hydroxybutyl (405.2 g, 528 mmol) in dichloromethane (20 mL) is addeddimethylaminopyridine (0.488 g, 4 mmol) and acetic anhydride (3.39 mL,36 mmol), and the mixture is stirred at room temperature for 3 hours.The mixture is diluted with methylene chloride, then washed with 5%aqueous sodium bicarbonate and brine and dried over Na₂SO₄. The residueis dried and recrystallized from acetonitrile to give the compound.

[0327] Step 2′: Dehydration at C-10,11 and derivation of C-12 to formintermediate Compound (9) from FIG. 4: R_(a) is —CH₂CH═CH₂, R_(c) andR_(e) are acetyl

[0328] To a sample of the compound from step 1′ (85.8 g, 100 mmol) indry THF (500 mL) cooled to −40° C. and flushed with nitrogen is addedsodium bis(trimethylsilyl)amide (125 mL, 125 mmol) over 20 minutes, andthe mixture is stirred at −40° C. for 40 minutes. To this mixture isadded a solution of carbonyldiimidazole (3.65 g, 22.56 mmol) in 5:3THF/DMF (800 mL) under nitrogen at −40° C. over 30 minutes, and themixture is stirred at −20° C. for 30 minutes. The mixture is stirred atroom temperature for 27 hours, then diluted with ethyl acetate. Themixture is washed with 5% sodium bicarbonate and brine, dried overNa₂SO₄, and concentrated to give the compound (9), which is takendirectly to the next step.

[0329] Using the procedures described in this example and schemes andmethods known in the synthetic organic chemistry art, the compounds ofFormula (1) wherein A, B, D and E are H can be prepared. In addition,using the preceding examples and the above-mentioned schemes and knownmethods, the compounds having the R_(a) substituent can be prepared aslisted: —CH₂CH═CH₂ —CH₂CH₂CH₃ —CH₂CH₂NH2, —CH₂CH═NOH. —CH₂CH₂CH₂OH —CH₂F—CH₂CH₂NHCH₂-phenyl —CH₂CH₂NHCH₂-(4-pyridyl) —CH₂CH₂NHCH₂-(4-quinolyl)—CH₂CH(OH)CN —CH(C(O)OCH₃)CH₂.-phenyl —CH₂CN —CH₂CH₂CH₂-phenyl—CH₂CH═CH-(4-fluorophenyl) —CH₂CH₂NHCH(CH₂-phenyl)—CH₂CH₂CH₂-(4-ethoxyphenyl) C(O)OH₃ —CH₂CH₂NHCH₂CH₂-(2- —CH₂CH₂NHCH₂CH₂-chlorophenyl) (2-chlorophenyl) —CH₂CH═CH-(4-chlorophenyl)—CH₂-(4-pyridyl) —CH₂CH═CH-(4-methoxyphenyl) —CH₂CH═CH-(4-pyridyl)—CH₂CH═CH-(3-quinoly)) —CH₂CH═CH-(4-quinolyl) —CH₂-phenyl—CH₂CH═CH-(5-quinolyl) —CH₂-(4-quinolyl) —CH₂CH═CH-(4-benzoxazolyl)—CH₂CH₂CH₂-(4-pyridyl) —CH₂CH═CH-(8-quinolyl) —CH₂CH₂CH₂-(4-quinolyl)—CH₂CH═CH-(1,3-dimethyl-2, 4-dioxo-5-pyrimidinyl)—CH₂CH₂CH₂-(5-quinolyl) —CH₂-CH═CH-(5- (3-isoxazolyl)-2-thiophenyl)—CH₂CH═CH-(4-benzimidazolyl) —CH₂-CH═CH═(5-(2-pyridyl)aminocarbonyl-2-furanyl)

Example 17 Preparation of Compound of Formula (1): A, B and E are H, Dis benzyl, R_(a) is allyl

[0330] Step 1: Preparation of 2-(R)-(BOC-amino)-3-pheny-1-propanol

[0331] To a 5.2 g (23.8 mmol) sample of di-t-butyl dicarbonate in 20 mLof methylene chloride held at 0° C. is added(R)-2-amino-3-phenyl-1-propanol (3.0 g, 19.8 mmol Aldrich), and thereaction mixture is stirred 1.5 hours at room temperature. The solventwas removed, and the residue is dried under high vacuum and takendirectly to the next step.

[0332] Step 2: Preparation of2-(R)-(BOC-amino)-1-O-methyl-anesulfonyloxy-3-phenylpropane

[0333] The material from step 1 is dissolved in 20 mL of methylenechloride and 5 mL of THF, and the solution is cooled to 0° C.Triethylamine (4.1 mL, 29.4 mmol) is added, then methanesulfonylchloride (1.9 mL, 24.5 mmol) is added slowly. The mixture is stirred 45minutes at room temperature, the the solvent is removed under vacuum.The residue is dissolved in ethyl acetate, and the solution is washedwith water and brine, dried (Na₂SO₄) and filtered. The solvent isremoved under vacuum to afford the title compound.

[0334] Step 3: Preparation of 1-azido-2-(R)-(BOC-amino)-3-phenylpropane

[0335] The compound from step 2 above (6.36 g, 193 mmol) is dissolved in25 mL of DMF, and 2.5 g (38 mmol) of NaN₃ is added. The reaction mixtureis stirred for 24 hours at 62° C. The solution is cooled at roomtemperature, then extracted with ethyl acetate. The organic extract iswashed with water and brine, dried (Na₂SO₄) and filtered. The solvent isremoved under vacuum to afford the title compound.

[0336] Step 4: Preparation of 1-azido-2-(R)-amino-3-phenylpropane

[0337] The compound from step 3 (4.3 g, 15.6 mmol) is dissolved in 30 mLof 4 N HCl in ethanol, and the reaction mixture is stirred for 1.5 hoursat room temperature. The solvent is stripped and chased with ether. Theresidue is dissolved in water, NaCl is added, and the mixture isextracted with ethyl ether, which is discarded. The aqueous layer isadjusted to pH 12 with K₂CO₃, saturated with NaCl, then extracted withCHCl₃. The organic extract is washed with brine, dried (Na₂SO₄) andfiltered. The solvent is removed under vacuum to afford the titlecompound.

[0338] Step 5: Preparation of 1,2-(R)-diamino-3-phenylpropane

[0339] A sample of the compound from step 4 (1.2 g, 6.8 mmol) ishydrogenated (4 atm) in ethanol over 1.2 g of 10% Pd/C for 21.5 hours atroom temperature. The mixture is filtered to remove the catalyst, andthe solvent is removed to afford the title compound.

[0340] Step 6: Form cyclic carbamate, compound (10) from FIG. 4: A, Band E are H, D is benzyl, R_(a) is allyl, R_(c) is benzoyl

[0341] The desired compound is prepared by stirring a solution ofcompound prepared as in Example 16, step 5, (which is the compound (7)from FIG. 5, wherein R_(a) is allyl, R_(c) is benzoyl), and1,2-(R)-diamino-3-phenylpropane, prepared as in step 5 above, in aqueousacetonitrile for an amount of time sufficient to consume substantiallyall of the starting material.

[0342] Step 7: Deprotection to Form Compound (10) from FIG. 4; A, B andE are H, D is benzyl, R_(a) is allyl, R_(c) is H

[0343] The title compound is prepared by deprotection of the compoundprepared in step 6 by heating in methanol according to the followingprocedure: a solution of the compound resulting from step 6 in methanol(20 mL) is stirred at reflux for 6 hours. The reaction mixture isconcentrated in vacuo and the residue is purified by chromatography onsilica gel (95:5:0.5 dichloromethane-methanol-ammonia) to give thedesired compound.

[0344] Step 8: Cyclization to Form Compound of Formula (1): A, B and Eare H, D is benzyl, R_(a) is allyl

[0345] The desired compound is prepared by heating a solution of thecompound prepared in step 7 in ethanol-acetic acid.

Example 18 Preparation of Compound of Formula (1): A is benzyl, B, D andE are H, R_(a) is allyl

[0346] Step 1: Form Cyclic Carbamate of Compound (10′) from FIG. 5; Abenzyl, B, D and E are H, Y is OH, R_(a) is allyl, R_(c) is benzoyl

[0347] The desired compound is prepared according to the method ofExample 17, step 7 except substituting (S)-2-amino-3-phenyl-1-propanol(Aldrich Chemical Co.) for 1,2-(R)-diamino-3-phenylpropane.

[0348] Step 2: Y Substitution to Form Compound (10′) from FIG. 5; A isbenzyl, B, D and E are H, Y is N₃, R_(a) is allyl, R_(c) is benzoyl

[0349] The desired compound is prepared by treating a solution in THF ofthe compound of step 1 with triphenylphosphine, diethylazodicarboxylate,and diphenylphosphorylazide.

[0350] Step 3: Deprotection to Form Compound (10′) from FIG. 5; A isbenzyl, B, D and E are H, Y is N₃, R_(a) is allyl, R_(c) is H

[0351] The desired compound is prepared by deprotection of the compoundprepared in step 2 by heating in methanol according to the procedure ofExample 17, step 7.

[0352] Step 4: Reduction to Form Compound (1) from FIG. 5, R_(a) isallyl

[0353] The desired compound is prepared by refluxing a solution in THFof the product of step 3 and triphenylphosphine.

[0354] Step 5: Form Compound of Formula (1) from FIG. 5: A is benzyl, B,D and E are H, R, is allyl.

[0355] The desired compound is prepared by heating a solution of thecompound prepared in step 4 in ethanol-acetic acid.

Example 19 Compound of Formula (1): A and E are phenyl, B and D and areH, R_(a) is allyl

[0356] The desired compound is prepared according to the method ofExample 17, steps 6-8, except substituting1,2-diphenyl-1,2-ethylenediamine (Aldrich Chemical Co.) for1,2-(R)-diamino-3-phenylpropane.

Example 20 Preparation of Compound of Formula (1): A is methyl, B, D andE are H, R_(a) is allyl

[0357] The desired compound is prepared according to the method ofExample 18, except substituting (S)-2-amino-1-propanol (Aldrich ChemicalCo.) for (S)-2-amino-3-phenyl-1-propanol.

Example 21 Preparation of Compound of Formula (1): A and D are methyl, Band E are H, R_(a) is allyl

[0358] Step 1: Preparation of meso-2,3-bis(methanesulfonyloxy)butane

[0359] Samples of meso-2,3-butanediol (10 g, 111 mmol, Aldrich) andtriethylamine (92.8 mL, 666 mmol) are dissolved in methylene chloride.The solution is cooled to −78° C., and methanesulfonyl chloride (25.8mL, 333 mmol) is added dropwise. A precipitate forms. The mixture isdiluted with additional methylene chloride, and the mixture is stirredfor 20 minutes at −78° C. and at 0° C. for 2 hours. The reaction mixtureis warmed to room temperature, diluted with additional solvent, andwashed with H₂O, aqueous NaHCO₃ and aqueous NaCl. The organic solutionis dried over MgSO₄, and the solvent is removed to afford the titlecompound.

[0360] Step 2: Preparation of meso-2,3-diazidobutane

[0361] A sample of the compound from step 1 (25 g) is dissolved in 250mL of DMF, and NaN₃ (40 g) is added. The mixture is stirred vigorouslyat 85° C. for 24 hours, then cooled to room temperature. The mixture isdiluted with 800 mL of ether, washed with H₂O, aqueous NaHCO₃ andaqueous NaCl, then dried over MgSO₄. The solution is filtered andconcentrated to afford the title compound.

[0362] Step 3: Preparation of meso-2-3-butanediamine

[0363] A sample of the compound from step 2 (13.0 g, 125 mmol) isdissolved in ethanol and hydrogenated at 4 atm over 10% Pd/C for 20hours at room temperature. The catalyst is removed by filtration, andthe solvent is removed under vacuum to afford the title compound.

[0364] Step 4: Preparation of Compound of Formula (1); A and D aremethyl B and E are H, R_(a) is allyl

[0365] The desired compound is prepared according to the method ofExample 17, steps 6-8, except substituting meso-2-3-butanediamine,prepared as in step 3, for the 1,2-(R)-diamino-3-phenylpropane thereof.

Example 22

[0366] Preparation of Compound of Formula (1): A and E taken together is—CH₂CH₂CH₂—, B and D are H, R_(a) is allyl

[0367] The desired compound is prepared according to the method ofExample 21, except substituting 1,2-cyclopentane diol (Aldrich ChemicalCo.) for meso 2,3-butanediol

Example 23 Compound of Formula (1): A, B, D, and E are H, R_(a) is—CH₂CH═CH-(3-quinolyl)

[0368] Step 1: Conversion of R_(a) to form compound (6), R_(a) is—CH₂CH═CH-(3-quinolyl), R_(c) is benzoyl

[0369] A mixture of the compound (6) (R_(a) is allyl) (1.80 g, 0.25mmol), palladium(II)acetate (11 mg, 0.05 mmol), and tri-o-tolylphosphine(30 mg, 0.10 mmol) and 3-bromoquinoline (68 μL, 0.5 mmol) inacetonitrile (2 mL) is cooled to −78° C., degassed, and sealed. Thereaction mixture is then warmed to 50° C. for 2 hours and stirred at 80°C. for 16 hours. The reaction mixture is taken up in ethyl acetate andwashed with aqueous 5% sodium carbonate, aqueous 2%tris(hydroxymethyl)aminomethane, and brine, dried over sodium sulfate,filtered, and concentrated in vacuo. Chromatography on silica gel (98:2dichloromethane-methanol) gives the title compound as an off-white foam.

[0370] Step 2: Deprotection to Form Compound of Formula (6); R_(a) is—CH₂CH═CH-(3-quinolyl), R_(c) is H

[0371] Deprotection of the compound prepared in step 1 is accomplishedby heating in methanol according to the procedure of Example 16, step 7.

[0372] Step 3: Conversion of R_(a)

[0373] The desired compound was prepared by coupling 3-bromoquinolinewith the product of Example 16, step 7.

[0374] Optional Step 4: Reduction to Form Compound of Formula (1): A, B,D, and E are H, R_(a) is —CH₂CH₂CH₂-(3-quinolyl)

[0375] To a sample of the compound from Example 23, step 3 (110 mg) inmethanol (10 mL) flushed with nitrogen is added 10% Pd/C (50 mg), andthe mixture is stirred at room temperature under 1 atm of hydrogen for16 hours. The mixture is filtered and concentrated, and the residue ispurified by chromatography on silica gel eluting with 95:5:0.5 to90:10:0.5 dichloromethane/methanol/dimethylamine to give the titlecompound.

[0376] Using the procedures described in the preceding examples andschemes and methods known in the synthetic organic chemistry art, thecompounds of Formulas (1)-(3) and (1′)-(3′) can be prepared wherein atleast one of A, B, D and E, and wherein two of which may be joined toform a ring are described below. ethyl pentenyl propyl hexenyl butylpyridyl pentyl pyrimidinyl hexyl pyridazinyl ethenyl indolyl propenylquinolyl butenyl naplithyl pentenyl quinoxalinyl hexynyl thienyl ethynylfuryl propynyl chromenyl butynyl

Example 24 Preparation of Compound of Formula (103) in FIG. 8: L is CO,T is NR, R_(a)=H, R_(c) is H

[0377] Step 1. Preparation of 6-hydroxy Analog of Compound (109)

[0378] A solution of 14,15-dehydroerythromycin A in methanol is cooledto −40° C. A stream of ozone is bubbled into the solution, and thereaction is vented through a trap containing an aqueous solution ofpotassium iodide. Ozone introduction is stopped when the brown color ofI₂ is noted in the trap solution, and the solution is purged with astream of nitrogen gas for 5 minutes. Dimethylsulfide is added, and themixture is allowed to warm slowly to ambient temperature to provide amethanolic solution of the 14-aldehyde. The concentration of thealdehyde can be determined by integration of the NMR signal for thealdehyde proton relative to that for the solvent methanol after dilutioninto CDCl₃.

[0379] The methanolic solution of the 14-aldehyde is cooled to 0 ° C.,then treated successively with 2 molar equivalents of acetic acid, 1.5equivalent of R-NH₂, and 5 equivalents of NaBH₃CN. The reaction ismonitored by thin-layer chromatography. When complete, the methanol isremoved under vacuum, and the crude residue is redissolved indichloromethane. This is cooled to 0 ° C., and 1 equivalent of phenylchloroformate is added. Triethylamine (3 equivalents) is added dropwise.After completion of carbamate formation, the mixture is poured intosaturated aqueous NaHCO₃ and extracted with dichloromethane. The extractis dried over MgSO₄, filtered, and evaporated. Chromatography yields the6-hydroxy analog of compound (109).

[0380] Step 2. Formation of Cyclic Carbamate Compound (112)

[0381] A solution of the compound in Step 1 in tetrahydrofuran is addeddropwise to a suspension of NaH (1.5 molar equivalents) intetrahydrofuran at 0 ° C. The reaction is monitored by thin-layerchromatography. When complete, saturated aqueous NH₄Cl is added, thetetrahydrofuran is removed under vacuum, and the mixture is extractedwith dichloromethane. The organic extracts are washed with 1 N NaOH toremove phenol, then with brine and dried over MgSO₄ prior toconcentration. The 6-hydroxy analog of compound (112)/(103) is isolatedby chromatography.

[0382] Using the procedures described in the preceding examples andschemes and methods known in the synthetic organic chemistry art, thecompounds of Formula (101) wherein L is CO and T is NH can be prepared.These compounds having the R_(a) substituent are described below:—CH₂CH═CH-phenyl —CH₂CH═CH-(3-quinolyl) —CH₂CH═CH₂ —CH₂CH₂CH₃—CH₂CH₂NH2, —CH₂CH═NOH. —CH₂CH₂CH₂OH —CH₂F —CH₂CH₂NHCH₂-phenyl—CH₂CH₂NHCH₂-(4-pyridyl) —CH₂CH₂NHCH₂-(4-quinolyl) —CH₂CH(OH)CN—CH(C(O)OCH₃)CH₂.-phenyl —CH₂CN —CH₂CH═CH-(4-chlorophenyl)—CH₂CH═CH-(4-fluorophenyl) —CH₂CH═CH-(4-methoxyphenyl)—CH₂CH₂CH_(2-(4-ethoxyphenyl)) —CH₂CH═CH-(3-quinoly))—CH₂CH₂NHCH₂CH₂-(2- chlorophenyl) —CH₂-phenyl —CH₂-(4-pyridyl)—CH₂-(4-quinolyl) —CH₂CH═CH-(4-pyridyl) —CH₂CH₂CH₂-(4-pyridyl)—CH₂CH═CH-(4-quinolyl) —CH₂CH₂CH₂-(4-quinolyl) —CH₂CH═CH-(5-quinolyl)—CH₂CH₂CH₂-(5-quinolyl) —CH₂CH═CH-(4-benzoxazolyl)—CH₂CH═CH-(4-benzimidazolyl) —CH₂CH═CH-(8-quinolyl)—CH₂—CH═CH-(5-(3-isoxazolyl)- —CH₂—CH═CH-(1,3-dimethyl- 2-thiophenyl)2,4-dioxo-5-pyrimidinyl) —CH₂-CH═CH═(5-(2-pyridyl)aminocarbonyl-2-furanyl)

[0383] Other Embodiments: Using the above procedures, compounds offormulas (101)-(103) can be formed wherein L is CO, T is: —N(CH₃);—NCH₂CH₂N(CH₃)₂; —N(CH₂CH═CH₂); —N(CH₂CH═CH-(3-quinolyl)); or —N(NH₂);

Example 25 Compound of Formula (103) in FIG. 8: L is CO, T is O, R_(a)is H, R_(c) is H

[0384] Step 1: Preparation of 6-hydroxy Analog of (109′)

[0385] A solution of 14,15-dehydroerythromycin A in methanol is cooledto −40° C. A stream of ozone is bubbled into the solution, and thereaction is vented through a trap containing an aqueous solution ofpotassium iodide. Ozone introduction is stopped when the brown color ofI₂ is noted in the trap solution, and the solution is purged with astream of nitrogen gas for 5 minutes. The solution is warmed to 0 ° C.,and a solution of sodium borohydride in isopropanol is added slowly. Thereaction is monitored by thin-layer chromatography. When complete, themethanol is removed under vacuum. The residue is redissolved indichloromethane, washed with aqueous ethylene glycol followed by brine,dried over MgSO₄, filtered, and evaporated. The compound is isolated bychromatography.

[0386] Step 2: Formation of the cyclic carbonate compound (112′)

[0387] A solution of the compound of Step 1 in dichloromethane istreated with phenyl chloroformate (2 molar equivalents) and4-dimethylaminopyridine (2 molar equivalents). After formation of theintermediate phenyl carbonate, the mixture is washed with water andbrine, then dried over MgSO₄, filtered, and evaporated. The crude phenylcarbonate is dissolved in tetrahydrofuran, and this solution is addeddropwise to a suspension of NaH (1.5 equivalents) in tetrahydrofuran at0° C. Upon completion of the cyclization, the reaction is quenched byaddition of saturated aqueous NH₄Cl, the tetrahydrofuran is removedunder vacuum, and the mixture is extracted with dichloromethane. Themixture is evaporated, redissolved in methanol and allowed to stand for24 hours, then evaporated and redissolved in dichloromethane. Thesolution is washed with 1 N NaOH to remove phenol, then washed withbrine and dried over MgSO₄ prior to concentration. The 6-hydroxy analogof compound (112′)/(103) is isolated by chromatography.

[0388] Using the procedures described in the preceding examples andschemes and methods known in the synthetic organic chemistry art, thecompounds of Formula (101) wherein L is CO and T is O can be prepared.These compounds include one of the R_(a) substituents listed below:—CH₂CH₂CH₃ —CH₂CH₂NH₂ —CH₂CH═NOH —CH₂CH₂CH₂OH —CH₂F —CH₂CH₂-phenyl—CH₂CH₂-(4-pyridyl) —CH₂CH₂-(4-quinolyl) —CH₂CH(OH)CN—CH(C(O)OCH₃)CH₂-phenyl —CH₂CN —CH₂CH═CH-(4-methoxyphenyl)—CH₂CH═CH-(4-fluorophenyl) —CH₂CH═CH-(8-quinolyl) —CH₂CH₂NHCH₂-phenyl—CH₂-phenyl —CH₂-(4-pyridyl) —CH₂-(4-quinolyl) —CH₂CH═CH-(4-pyridyl)—CH₂CH₂CH₂-(4-pyridyl) —CH₂CH═CH-(4-quinolyl) —CH₂CH₂CH₂-(4-quinolyl)—CH₂CH═CH-(5-quinolyl) —CH₂CH₂CH₂-(5-quinolyl)—CH₂CH═CH-(4-benzoxazolyl) —CH₂CH═CH-(4-benzimidazolyl)

Example 26 Preparation of Compounds (103) and (101) in FIG. 11 a and 11b

[0389] Step 1: Protection of 9 Keto Group to Form Oxime

[0390] To a solution of the compound (I_(a)), which is the 6-hydroxyanalog of compound (103), prepared in either Example 24 or 25 inpropanol is added aqueous hydroxylamine and acetic acid, and stirredunder appropriate conditions. The reaction is stopped and the solvent isremoved to yield compound (I_(b)).

[0391] Step 2: Prepare Derivitized Oxime

[0392] To a solution of the compound prepared in Step 1 anddichloromethane, is added 1,1 diisopropoxycyclohexane under appropriateconditions with pyrH⁺OTs⁻. The reaction mixture is stirred underappropriate conditions, is stopped, washed and dried. The solvent isremoved and the excess 1,1-diisopropoxycyclohexane is removed to yieldcompound (I_(c)).

[0393] Step 3: Protection of 2′ and 4″ Hydroxyl Groups

[0394] To a solution of the compound prepared in Step 2 indichloromethane is added 1-trimethylsilylimidazole andchlorotrimethylsilane in dichloromethane. The mixture is stirred underappropriate conditions. The reaction is stopped, filtered and washed.The solvent is removed and the compound (I_(d)) purified.

[0395] Step 4: Alkylation of 6-OH to form protected form of compound(103)

[0396] A solution of the compound prepared in Step 3 indimethylsulfoxide/tetrahydrofuran (DMSO/THF) is treated with a solutionof allyl bromide under appropriate conditions. A mixture of potassiumt-butoxide in THF and DMSO is added gradually. The reaction is diluted,washed and dried. Filtration yields a crude product of compound (103).

[0397] Step 5: Deprotection of 9 oximes 2′ and 4″ Hydroxyl Groups

[0398] The compound prepared in Step 4, acetic acid, and acetonitrile inwater is stirred under appropriate conditions. The mixture is dried anddirectly used in the next step.

[0399] Step 6: Deoximation to Form Compound (103) having 9 Keto Group.

[0400] A mixture of the compound prepared in Step 5, water, and sodiumhydrosulfite is kept under appropriate conditions. The alkalinity of themixture is increased and the reaction extracted. The extract is washed,dried, filtered and concentrated to yield a crude product of compound(103).

[0401] Step 7: Removal of Cladinose

[0402] To a suspension of the compound prepared in Step 6 in ethanol, isadded HCl gradually under appropriate conditions and additional HCl isadded. The mixture is stirred for 18 hours, cooled and the alkanity isincreased. Filtration yields the compound (I_(e)).

[0403] Step 8: Protection of 2′ Hydroxyl Group

[0404] To a solution of the compound prepared in Step 7 indichloromethane is added Bz₂O. The mixture is stirred under appropriateconditions to yield the compound (I_(f)).

[0405] Step 9: Conversion of 3-OH to 3-carbonyl

[0406] To a solution under nitrogen of N-chloro succinimide indichloromethane is gradually added dimethylsulfide. The resulting slurryis stirred and a solution of the compound prepared in step 8 indichloromethane is added and mixed. Triethylamine is added dropwise andthe reaction mixture stirred under appropriate conditions. The reactionmixture is worked up to give compound (101) of the invention.

[0407] Step 10: —OR_(a) Conversions

[0408] To a solution under nitrogen of the 2′ protected compoundprepared in Step 9, palladium (II) acetate, and P(o-tolyl)₃ is addedhalogen-quinolyl and triethylamine and the mixture is stirred underappropriate conditions. The reaction mixture is worked up to givecompound (101) having the R_(a) position converted from allyl toquinolyl.

[0409] Step 11: Deprotection of the 2′ Hydroxyl Group

[0410] To a solution of the compound prepared in Step 10 is addedmethanol and the mixture is stirred under appropriate conditions. Thesolvent is removed in vacuo to give compound (101).

Example 27 Conversion at —OR_(a)

[0411] A. Allyl→—CH₂CHO

[0412] The compound from Example 10 (14.0 g) is dissolved in CH₂Cl₂ (200mL) and the solution is cooled to −78° C. under a nitrogen atmosphere.Ozone is then bubbled through the solution until a blue color persisted.The reaction is then purged with N₂ until colorless and dimethylsulfide(14 mL) is added, and the reaction mixture is warmed to 0° C. Afterstirring for 90 min, the reaction mixture is concentrated under reducedpressure to give a light-yellow foam. This material is dissolved in THF(300 mL) and treated with triphenylphosphine (8 g) at reflux for 6hours, then the reaction mixture is concentrated under reduced pressure.Chromatography (1:1 acetone/hexanes to 3:1 acetone/hexanes with 0.5%TEA) gave the product.

[0413] B. —CH₂CHO→—CH₂CH₂NHCH₂Phenyl

[0414] The compound from Example 11A (120 mg, 0.187 mmol) andbenzylamine (40 μL, 0.366 mmol, 2 equiv) are dissolved in 3 mL of drydichloromethane. Molecular sieves (4 Å) are added and the reaction isstirred overnight. The reaction is then filtered and concentrated underreduced pressure. The resulting imine is dissolved in MeOH (5 mL), acatalytic amount of 10% Pd on carbon is added, and the reaction isstirred rapidly under 1 atm of H₂ pressure for 20 hours. The mixture isthen filtered through a Celite pad, and the solution concentrated underreduced pressure. Chromatography (SiO₂, 5% MeOH/dichloromethane with0.2% NH₄OH) gives the desired material (84 mg) as a white solid.

[0415] C. —CH₂CHO→—CH₂CH₂NHCH₂Phenyl

[0416] This compound is prepared from the compound of Example 11A (108mg, 0.169 mmol) and phenethylamine (42 μL, 0.334 mmol, 2 equiv) usingthe procedure described for Example 11B. Chromatography (SiO₂, 5%MeOH/dichloromethane with 0.5% NH₄OH) gives the desired material.

[0417] D. —CH₂CHO→—CH₂CH₂NHCH₂CH₂CH₂Phenyl

[0418] This compound is prepared from the compound of Example 11A (100mg, 0.156 mmol) and 3-phenyl-1-propylamine (40 μL, 0.282 mmol, 1.8equiv) using the procedure described for Example 11B. Chromatography(SiO₂, 5% MeOH/dichloromethane with 0.5% NH₄OH) gives the desiredmaterial.

[0419] E. —CH₂CHO→—CH₂CH₂NHCH₂CH₂CH₂CH₂Phenyl

[0420] This compound is prepared from the compound of Example 11A (170mg, 0.266 mmol) and 4-phenyl-1-butylamine (68 μL, 0.431 mmol, 1.6 equiv)using the procedure described for Example 11B. Chromatography (SiO₂, 5%MeOH/dichloromethane with 0.2% NH₄OH) gives the desired material.

[0421] F. —CH₂CHO→—CH₂CH₂NHCH₂CH₂CH₂-(3-quinolyl)

[0422] The compound from Example 11A (135 mg, 0.211 mmol) and3-(3-quinolyl)-1-propylamine (70 mg, 0.376 mmol, 1.8 equiv) aredissolved in 4 mL of dry dichloromethane. Molecular sieves (4 Å) areadded and the reaction is stirred overnight. The reaction is thenfiltered and concentrated under reduced pressure. The resulting imine isdissolved in MeOH (5 mL) and treated with NaCNBH₃ (about 100 mg) andenough AcOH to turn bromocresol green indicator from blue to yellow.After stirring for 4 hours, the reaction mixture is poured intosaturated NaHCO₃ solution and extracted into dichloromethane. Theorganic portion is washed with saturated NaHCO₃, H₂O and brine, dried(Na₂SO₄) and concentrated under reduced pressure. Chromatography (SiO₂,5% MeOH/dichloromethane with 0.5% NH₄OH to 10% MeOH/dichloromethane with1% NH₄OH) gives the desired material.

[0423] G. —CH₂CHO→—CH₂CH₂NHCH₂(3-quinolyl)

[0424] The title compound is prepared from the compound of Example 11A(150 mg, 0.234 mmol) and 3-(aminomethyl)quinoline (100 mg, 0.633 mmol,2.7 equiv) using the procedure described for Example 11F. Chromatography(SiO₂, 5% MeOH/dichloromethane with 0.5% NH₄OH) gives the desiredmaterial.

[0425] The 3-(aminomethyl)quinoline reagent is prepared by methods knownin the art.

[0426] Other embodiments of the formulas (101)-(103) wherein R_(b) is H,R_(c) is H, L is —CO—, T is —NH—, and R_(d)′ is methyl, ethyl, propyl,butyl, benzyl, vinyl, or 3-hydroxy butyl are those wherein R_(a) isconverted from —CH₂CHO to: —CH₂CH₂NHCH₂(6-quinolyl); —CH₂CH═NO(phenyl);—CH₂CH═NOCH₂(phenyl); —CH₂CH═NOCH₂(4-NO₂-phenyl);—CH₂CH═NOCH₂(4-quinolyl); —CH₂CH═NOCH₂(2-quinolyl);—CH₂CH═NOCH₂(3-quinolyl); —CH₂CH═NOCH₂(6-quinolyl);—CH₂CH═NOCH₂(1-naphthyl); —CH₂CH═NOCH₂(2-naphthyl);—CH₂CH₂NHOCH₂(phenyl); —CH₂CH₂NHOCH₂(4-NO₂-phenyl); —CH₂C(O)-phenyl;—CH₂C(O)-(4-F-phenyl); —CH₂CH═NNHC(O)phenyl; or —CH₂CH(OH)-phenyl.

[0427] H. —CH₂CH═CH-(2-quinolyl)→—CH₂CH₂CH₂(2-quinolyl)

[0428] A mixture of the compound from Example 10 where R_(a) is—CH₂CH═CH-(2-quinolyl) (230 mg) and 10% Pd/C (50 mg) in 30 mL ofmethanol and 15 mL of ethyl acetate is flushed with nitrogen and stirredunder 1 atm of hydrogen at room temperature for 22 hours. The mixture isfiltered, and the filtrate is concentrated under reduced pressure.Chromatography on silica gel (5% MeOH/dichloromethane with 0.5% NH₄OH)gives the desired material.

[0429] I. —CH₂CH═CH-(3-quinolyl)→—CH₂(2-(3-quinolyl)cyclopropyl)

[0430] To a solution of diazomethane (0.64 M, 3.12 mL, 2.00 mmol) inether is added a solution of the compound from Example 10 wherein R_(a)is —CH₂CH═CH-(2-quinolyl) (153 mg, 0.200 mmol) in dichloromethane (5.0mL) at 0° C. under nitrogen. A small amount (2 mg) of palladium acetateis added, and the mixture is stirred for 20 minutes. Another portion ofdiazomethane (3 mL) is added, and the mixture is stirred for anotherhour. The solvents are evaporated, and the residue is purified bychromatography on silica gel (5% MeOH/dichloromethane with 0.5% NH₄OH)to give the title compound as a white solid.

Example 28 Conversions at R_(c)

[0431] A. —H→propanoyl (R_(a) is —CH₂CH═CH-(2-quinolyl))

[0432] To a solution of the compound from Example 10 converted at R_(a),wherein R_(a) is —CH₂CH═CH-(2-quinolyl), (152 mg) in dichloromethane isadded propionic anhydride (52 μL) and triethylamine (56 μL), and themixture is stirred for 24 hours at room temperature. The mixture isdiluted with ethyl acetate, and this is washed with 5% NaHCO₃ solutionand brine, dried (Na₂SO₄) and concentrated under reduced pressure. Theresidue is chromatographed on silica gel (1:1 acetone/hexanes) to givethe title compound as a white foam.

[0433] B. —H→—ethylsuccinoyl (R_(a) is —CH₂CH═CH-(2-quinolyl))

[0434] To a solution of the compound from Example 10 converted at R_(a),wherein R_(a) is —CH₂CH═CH-(2-quinolyl) (153 mg, 0.200 mmol) indichloromethane (10 mL) at 0° C. is added ethyl succinyl chloride (29μL) and triethylamine (56 μL), and the mixture is stirred for 24 hoursat room temperature. The mixture is diluted with ethyl acetate, and thisis washed with 5% NaHCO₃ solution and brine, dried (Na₂SO₄) andconcentrated under reduced pressure. The residue is chromatographed onsilica gel (1:1 acetone/hexanes) to give the title compound as a whitefoam.

[0435] Further variations and modifications of the foregoing will beapparent to those skilled in the art and are intended to be encompassedby the claims appended hereto.

Example 29 Fluorination of C2 Position Before Fused Ring FormationSynthesis of2′-O-benzoyl-6-O-propargyl-3-descladinosyl-3-oxo-10,11-anhydro-2-fluoro-15-methylerythromycinA

[0436] A solution of2′-O-benzoyl-6-O-propargyl-3-descladinosyl-3-oxo-10,11-anhydro-15-methyl-erythromycinA in tetrahydrofaran under inert atmosphere is cooled to −78° C. andtreated with 1.0 M potassium tert-butoxide in tetrahydrofuran. Themixture is stirred for 5 minutes, and a solution ofN-fluorobenzenesulfonimide in tetrahydrofuran is added in three portionsover 2 hours. After addition, the reaction is allowed to warm to ambienttemperature and kept for an additional 5 hours. Aqueous K₂CO₃ is added,and the mixture is extracted with CH₂Cl₂. The organic extracts arecombined, dried over MgSO₄, filtered, and evaporated. Chromatography onsilica gel gives the product.

Example 30 Derivatization of C-13 Position for Intermediate Compounds ofCompounds (1)-(3) or (1′)-(3′) Starting Material: 15-Aminoerythromycin ADiacetate Salt

[0437]

[0438] A solution of 15-azidoerythromycin A (7.75 g, 10 mmol) in 50 mLof methanol is treated with acetic acid (2.0 mL) and 10% palladium oncarbon (0.1 g) and stirred under 1 atm of hydrogen gas until thin-layerchromatographic analysis reveals complete reduction of the startingmaterial. The suspension is filtered through Celite to remove thecatalyst, then evaporated to dryness to yield the product, which is usedas a starting material for the following derivatizations.

[0439] A. Synthesis of 15-(quinol-4-ylacetamido)erythromycin A

[0440] A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10mL of dichloromethane is treated sequentially with quinol-4-ylacetylchloride (350 mg) and triethylamine (0.5 mL) at 0° C. After 3 hours, thereaction is diluted with dichloromethane and washed three times withsaturated aqueous NaHCO₃. The organic phase is dried over MgSO₄,filtered, and evaporated to yield the crude product. Purification bysilica gel chromatography yields the pure product.

[0441] B. Synthesis of 15-(3-(quinol-4-yl)propionamido)erythromycin A

[0442] A solution of 15-Aminoerythromycin A diacetate salt (1.0 g) in 10mL of dichloromethane is treated sequentially with3-(quinol-4-yl)propionyl chloride (400 mg) and triethylamine (0.5 mL) at0° C. After 3 hours, the reaction is diluted with dichloromethane andwashed three times with saturated aqueous NaHCO₃. The organic phase isdried over MgSO₄, filtered, and evaporated to yield the crude product.Purification by silica gel chromatography yields the pure product.

[0443] C. Synthesis of 15-(isoquinol-4-ylacetamido)erythromycin A

[0444] A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10mL of dichloromethane is treated sequentially with isoquinol-4-ylacetylchloride (350 mg) and triethylamine (0.5 mL) at 0° C. After 3 hours, thereaction is diluted with dichloromethane and washed three times withsaturated aqueous NaHCO₃. The organic phase is dried over MgSO₄,filtered, and evaporated to yield the crude product. Purification bysilica gel chromatography yields the pure product.

[0445] D. Synthesis of 15-(3-(isoguinol-4-yl)propionamido)erythromycin A

[0446] A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10mL of dichloromethane is treated sequentially with3-(isoquinol-4-yl)propionyl chloride (400 mg) and triethylamine (0.5 mL)at 0° C. After 3 hours, the reaction is diluted with dichloromethane andwashed three times with saturated aqueous NaHCO₃. The organic phase isdried over MgSO₄, filtered, and evaporated to yield the crude product.Purification by silica gel chromatography yields the pure product.

[0447] E. Synthesis of 15-((quinol-5-ylamino)acetamido)erythomcin A

[0448] A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10mL of dichloromethane is treated sequentially with(quinol-5-ylamino)acetic acid (0.30 g), dicyclohexylcarbodiimide (0.4g), 1-hydroxybenzotriazole (0.25 g), and triethylamine (0.5 mL) at 0° C.After 3 hours, the reaction is diluted with dichloromethane and washedthree times with saturated aqueous NaHCO₃. The organic phase is driedover MgSO₄, filtered, and evaporated to yield the crude product.Purification by silica gel chromatography yields the pure product.

[0449] F. Synthesis of 15-((quinol-6-ylamino)acetamido)erythromycin A

[0450] A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10mL of dichloromethane is treated sequentially with(quinol-6-ylamino)acetic acid (0.30 g), dicyclohexylcarbodiimide (0.4g), 1-hydroxybenzotriazole (0.25 g), and triethylamine (0.5 mL) at 0° C.After 3 hours, the reaction is diluted with dichloromethane and washedthree times with saturated aqueous NaHCO₃. The organic phase is driedover MgSO₄, filtered, and evaporated to yield the crude product.Purification by silica gel chromatography yields the pure product.

[0451] G. Synthesis of15-((quinol-4-ylmethyl)carbamoylamino)erythromycin A

[0452] A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10mL of dichloromethane is treated sequentially withquinoline-4-methoxycarbonyl chloride (400 mg) and triethylamine (0.5 mL)at 0° C. After 3 hours, the reaction is diluted with dichloromethane andwashed three times with saturated aqueous NaHCO₃. The organic phase isdried over MgSO₄, filtered, and evaporated to yield the crude product.Purification by silica gel chromatography yields the pure product.

1. A compound of the formula

wherein R_(a) is substituted or unsubstituted alkyl (1-10C); substitutedor unsubstituted alkenyl (2-10C); substituted or unsubstituted alkynyl(2-10C); substituted or unsubstituted aryl (3-20C); substituted orunsubstituted arylalkyl (4-20C); or OR_(a) is replaced by H; R_(b) is Hor halogen; R_(c) is H or a protecting group; R_(d) is methyl,unsubstituted alkyl (3-10C); substituted alkyl (1-10C); substituted orunsubstituted alkenyl (2-10C); substituted or unsubstituted alkynyl(2-10C); substituted or unsubstituted aryl (3-20C); or substituted orunsubstituted arylalkyl (4-20C); substituted or unsubstitutedarylalkenyl (5-20C); substituted or unsubstituted arylalkynyl (5-20C);substituted or unsubstituted amidoarylalkyl (5-20C); substituted orunsubstituted amidoarylalkenyl (5-20C); or substituted or unsubstitutedamidoarylalkynyl (5-20C) R_(e) is H or a protecting group; each of A, B,D and E is independently H, substituted or unsubstituted alkyl (1-10C)wherein any pair of said A, B, D and E forms a 3-7-membered ringoptionally containing one or more heteroatoms, with the proviso that atleast two of said A, B, D and E must be hydrogen; including anypharmaceutically acceptable salts thereof and any stereoisomeric formsand mixtures of stereoisomeric forms thereof.
 2. The compound of claim 1wherein R_(d) is methyl, propyl or vinyl.
 3. The compound of claim 1wherein R_(a) is arylalkenyl or arylalkynyl.
 4. The compound of claim 3wherein R_(a) is 3-aryl prop-2-enyl or 3-aryl prop-2-ynyl.
 5. Thecompound of claim 4 wherein said aryl is 3-quinolyl, 4-quinolyl or5-quinolyl, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-methoxyphenyl,6-quinolyl, 6-quinoxalyl, 6-amino-3-quinolyl, or 4-isoquinolyl.
 6. Thecompound of claim 1 wherein R_(a) is H or lower C1-C3 alkyl.
 7. Thecompound of claim 6 wherein R_(a) is methyl.
 8. The compound of claim 1wherein R_(b) is fluoro.
 9. A pharmaceutical composition comprising thecompound of claim 1 in admixture with a pharmaceutically acceptableexcipient.
 10. A method to control infection in a subject which methodcomprises administering to a subject in need of such control aneffective amount of the compound of claim 1 or a pharmaceuticalcomposition thereof.
 11. A method to preserve material from microbialdecay which method comprises providing said material with an effectiveamount of the compound of claim
 1. 12. A compound of the formula

wherein R_(a) is substituted or unsubstituted alkyl (1-10C); substitutedor unsubstituted alkenyl (2-10C); substituted or unsubstituted alkynyl(2-10C); substituted or unsubstituted aryl; substituted or unsubstitutedarylalkyl; or OR_(a) is replaced by H; R_(b) is H or halogen; R_(c) is Hor a protecting group; R_(d)′ is H, substituted or unsubstituted alkyl(1-10C); substituted or unsubstituted alkenyl (2-10C); substituted orunsubstituted alkynyl (2-10C); substituted or unsubstituted aryl(3-10C); substituted or unsubstituted arylalkyl (4-10C); substituted orunsubstituted arylalkynyl (5-20C); substituted or unsubstitutedamidoarylalkyl (5-20C); substituted or unsubstituted amidoarylalkenyl(5-20C); or substituted or unsubstituted amidoarylalkynyl (5-20C); R_(e)is H or a protecting group; L is methylene or carbonyl; T is —O—,—N(R)—, or —N(OR)—, —N(NHCOR)—, —N(N═CHR)—, or —N(NHR)— wherein R is Hor R_(a) as defined above, with the proviso that when L is methylene, Tis —O—; one of Z and Y is H and the other is OH, protected OH, or amino,mono- or dialkylamino, protected amino, or an amino heterocycle or Z andY together are ═O, ═NOH or a derivatized oxime; including anypharmaceutically acceptable salts thereof and any stereoisomeric formsand mixtures of stereoisomeric forms thereof.
 13. The compound of claim12 wherein R_(d)′ is H, methyl, ethyl, or propyl.
 14. The compound ofclaim 12 wherein R_(a) is arylalkenyl or arylalkynyl.
 15. The compoundof claim 14 wherein R_(a) is 3-aryl prop-2-enyl or 3-aryl prop-2-ynyl.16. The compound of claim 15 wherein said aryl is 3-quinolyl, 4-quinolylor 5-quinolyl, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-methoxyphenyl,6-quinolyl, 6-quinoxalyl, 6-amino-3-quinolyl, or 4-isoquinolyl.
 17. Thecompound of claim 12 wherein R_(a) is H or lower C1-C3 alkyl.
 18. Thecompound of claim 18 wherein R_(a) is methyl.
 19. The compound of claim12 wherein R_(b) is fluoro.
 20. A pharmaceutical composition comprisingthe compound of claim 12 in admixture with a pharmaceutically acceptableexcipient.
 21. A method to control infection in a subject which methodcomprises administering to a subject in need of such control aneffective amount of the compound of claim 12 or a pharmaceuticalcomposition thereof.
 22. A method to preserve material from microbialdecay which method comprises providing said material with an effectiveamount of the compound of claim 12.